CN105121661B - The method of phase is determined for genome assembling and haplotype - Google Patents

Abstract

The present invention provides for greatly speeding up and improve the method for from the beginning genome assembling.Method disclosed herein utilizes data analysing method, makes the from the beginning assembling of the genome from one or more subjects quick and cheap.The present invention further provides method disclosed herein can be used for a variety of applications, including the fixed phase of haplotype and macro genome analysis.

Description

Methods for genome assembly and haplotype phasing

Cross References to Related Applications

This application claims the benefit of Provisional Application No. 61/759,941, filed February 1, 2013, and Provisional Application No. 61/892,355, filed October 17, 2013, the disclosures of which are incorporated herein by reference.

technical field

The present invention provides genome assembly and haplotype phasing methods for identifying short, medium and long junctions within the genome.

Background technique

Theoretically and practically, it is still difficult to generate high-quality, highly contiguous genome sequences.

Contents of the invention

A permanence limitation of next-generation sequencing (NGS) data is the inability to span large genomic repetitive regions due to short reads and relatively small insert sizes. This defect significantly affects de novo assembly. Because the nature and arrangement of genomic rearrangements are uncertain, contigs separated by long repeat regions cannot be joined or resequenced. Furthermore, phasing information is difficult to determine because variants cannot be confidently associated with haplotypes over long distances. The present invention addresses all of these issues simultaneously by generating extremely long-range read pairs (XLRPs) spanning genomic distances in the hundreds of thousands of bases and up to megabases with appropriate input DNA . These data are invaluable for overcoming the problems caused by large repetitive regions in the genome (including centromeres); saving the cost of de novo assembly; and generating resequencing data with sufficient completeness and accuracy for individualized medicine .

It is important to use remodeled chromatin in the process of forming associations between very distant but molecularly connected DNA segments. The present invention enables distant fragments to be brought together and covalently joined through chromatin conformation, thereby physically linking previously distant parts of the DNA molecule. Subsequent processing enables the sequence of associated fragments to be determined, generating read pairs whose spacing on the genome extends the full length of the input DNA molecule. Since read pairs originate from the same molecule, these read pairs also contain phase information.

In some embodiments, the present invention provides methods capable of producing high-quality assemblies with less data than previously required. For example, the methods disclosed herein provide genome assemblies from only two lanes of Illumina HiSeq data.

In other embodiments, the present invention provides methods capable of generating chromosome-level phasing using a long-range read pair approach. For example, the methods disclosed herein can phase 90% or more of the heterozygous single nucleotide polymorphisms (SNPs) for the individual with an accuracy of at least 99% or greater. This accuracy is comparable to phasing produced by substantially more expensive and laborious methods.

In some embodiments, methods capable of generating genomic DNA fragments on the megabase scale can be used in conjunction with the methods disclosed herein. Long DNA fragments can be generated to confirm the ability of the method to generate read pairs spanning the longest fragments afforded by those extractions. In some cases, DNA fragments longer than 150 kbp were extracted and used to generate XLRP libraries.

The present invention provides methods for greatly speeding up and improving de novo genome assembly. The methods disclosed herein utilize data analysis methods that allow rapid, inexpensive de novo assembly of genomes from one or more subjects. The present invention further provides that the methods disclosed herein can be used in a variety of applications, including haplotype phasing and metagenomic analysis.

In certain embodiments, the invention provides a method for genome assembly comprising the steps of: generating multiple contigs; generating multiple contigs from data generated by probing chromosomes, chromatin, or reconstructing the physical layout of chromatin; read pairs; mapping or assembling the plurality of read pairs into the plurality of contigs; using the read mapping or assembly data to construct an adjacency matrix of the contigs; and analyzing the adjacency matrix to determine the adjacency matrix through the contigs Path, which represents the order of contigs and/or orientation to the genome. In a further embodiment, the invention provides that at least about 90% of the read pairs are weighted to include information about which read pairs represent short-range contacts and which read pairs represent long-range contacts by taking a function of the distance of each read to the edge of the contig contact information. In other embodiments, the adjacency matrix is rescaled to reduce the weight of substantial contacts on some contigs representing promiscuous regions of the genome, such as conserved binding sites for one or more mediators of backbone interactions that regulate chromatin, Such as the transcriptional repressor CTCF. In other embodiments, the present invention provides methods for genome assembly of a human subject, wherein a plurality of contigs are generated from the human subject's DNA, and wherein by analyzing the human subject's chromosomes, chromatin, or Multiple read pairs were generated from reconstituted chromatin made from naked DNA from human subjects.

In a further embodiment, the invention provides that multiple contigs are generated by using shotgun sequencing, the method comprising: fragmenting long stretches of subject DNA into random fragments of indeterminate size; The sequencing method sequences the fragments to generate a plurality of sequencing reads; and assembles the sequencing reads to form a plurality of contigs.

In certain embodiments, the invention provides that multiple read pairs are generated by probing the physical layout of chromosomes, chromatin, or remodeling chromatin using Hi-C based techniques. In a further embodiment, the Hi-C-based technique comprises: crosslinking chromosomes, chromatin, or remodeled chromatin with a fixative, such as formaldehyde, to form DNA-protein crosslinks; Cross-linked DNA-proteins are cleaved by multiple restriction enzymes to generate multiple DNA-protein complexes containing sticky ends; the stickiness is filled in with nucleotides containing one or more markers end, the label, such as biotin, to produce blunt ends that are then ligated together; fragment the multiple DNA-protein complexes; fragments of the junctions; and sequencing the fragments containing the junctions using high-throughput sequencing to generate multiple read pairs. In further embodiments, multiple read pairs for use in the methods disclosed herein are generated from data generated by probing the physical layout of remodeled chromatin.

In various embodiments, the present invention provides for the determination of multiple read pairs by probing the physical arrangement of chromosomes or chromatin isolated from cultured cells or primary tissues. In other embodiments, multiple read pairs can be determined by probing the physical layout of the remodeled chromatin by combining naked DNA obtained from one or more subject samples with the isolated group protein complexes.

In other embodiments, the present invention provides a method of determining haplotype phasing comprising the step of: identifying one or more heterozygous sites in a plurality of read pairs, wherein the read pairs containing double heterozygous Read pairs for loci determined for phased data for allelic variants.

In various embodiments, the present invention provides a method for high-throughput bacterial genome assembly comprising the steps of generating Multiple read pairs; this modified Hi-C-based method includes the steps of: collecting the microorganism from the environment; adding a fixative, such as formaldehyde, to form crosslinks in each microbial cell, wherein the read pairs map to different overlapping Groups indicate which contigs are from the same species.

In some embodiments, the invention provides a method for genome assembly comprising: (a) generating a plurality of contigs; (b) generating contigs from chromosomes, chromatin, or remodeling the physical layout of chromatin (c) map the plurality of read pairs to the plurality of contigs; (d) construct an adjacency matrix of the contigs using the read mapping data; and (e) analyze the adjacency matrix to determine a path through the contigs that represents the order of the contigs and/or the orientation to the genome.

In a further embodiment, the present invention provides a method of generating multiple read pairs by probing the physical layout of chromosomes, chromatin, or remodeling chromatin using Hi-C based techniques. In a further embodiment, the Hi-C-based technique comprises: (a) cross-linking chromosomes, chromatin, or remodeled chromatin with a fixative to form DNA-protein cross-links; (b) using one or more A restriction endonuclease cleaves the cross-linked DNA-protein to generate multiple DNA-protein complexes containing cohesive ends; (c) filling in the cohesive ends with nucleotides containing one or more labels , to generate blunt ends, which are then ligated together; (d) shearing the plurality of DNA-protein complexes into fragments; (e) by using the one or more markers, pulling and (f) sequencing the junction-containing fragments using a high-throughput sequencing method to generate multiple read pairs.

In certain embodiments, multiple read pairs are determined by probing the physical arrangement of chromosomes or chromatin isolated from cultured cells or primary tissues. In other embodiments, multiple read pairs are determined by probing the physical layout of remodeled chromatin by complexing naked DNA obtained from one or more subject samples with isolated histones Forming.

In some embodiments, the weighting is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% or more by taking a function of the distance of the reads from the edge of the contig The plurality of read pairs is increased to reflect a higher probability of short contacts than long contacts. In some embodiments, the adjacency matrix is rescaled to reduce the weight of a large number of contacts on some contigs that represent promiscuous regions of the genome.

In certain embodiments, the genomic promiscuous region includes one or more conserved binding sites for one or more mediators that regulate chromatin backbone interactions. In some embodiments, the mediator is the transcriptional repressor CTCF.

In some embodiments, the present invention provides methods for genome assembly of a human subject, wherein a plurality of contigs are generated from the DNA of the human subject, and wherein the human subject's chromosomes, chromatin, or Multiple read pairs were generated from reconstituted chromatin made from naked DNA from the subject.

In other embodiments, the present invention provides methods for determining haplotype phasing comprising: identifying one or more heterozygous sites in a plurality of read pairs, wherein one or more heterozygous sites can be identified by identifying read pairs determined for phased data for allelic variants.

In other embodiments, the present invention provides a method for metagenomic assembly wherein multiple read pairs are generated by probing the physical layout of multiple microbial chromosomes using a modified Hi-C-based method; the modified The Hi-C-based method involves the following steps: collection of microorganisms from the environment; addition of a fixative, such as formaldehyde, to form crosslinks in each microbial cell, and wherein mapping of read pairs to different contigs indicates which contigs are from the same species.

In some embodiments, the invention provides a method of assembling multiple contigs derived from a single DNA molecule, comprising: generating multiple read pairs from a single DNA molecule, and assembling the contig using the read pairs, wherein At least 1% of read pairs span a distance greater than 50 kB on the single DNA molecule, and the read pairs are generated within 14 days. In some embodiments, at least 10% of read pairs span a distance greater than 50 kB on said single DNA molecule. In other embodiments, at least 1% of read pairs span a distance greater than 100 kB on said single DNA molecule. In a further embodiment, the read pairs are generated within 7 days.

In other embodiments, the present invention provides a method of assembling multiple contigs derived from a single DNA molecule, comprising: generating multiple read pairs from a single DNA molecule, and using the read pairs to assemble contigs, wherein at least 1% of read pairs span a distance greater than 30 kB on that single DNA molecule. In some embodiments, at least 10% of read pairs span a distance greater than 30 kB on said single DNA molecule. In other embodiments, at least 1% of read pairs span a distance greater than 50 kB on said single DNA molecule.

In other embodiments, the present invention provides methods for haplotype phasing comprising generating multiple read pairs from a single DNA molecule and using said read pairs to assemble multiple contigs of said DNA molecule, wherein at least 1 % of read pairs span a distance greater than 50 kB on the single DNA molecule and are haplotype phased with greater than 70% accuracy. In some embodiments, at least 10% of read pairs span a distance greater than 50 kB on said single DNA molecule. In other embodiments, at least 1% of read pairs span a distance greater than 100 kB on said single DNA molecule. In a further embodiment, haplotype phasing is performed with greater than 90% accuracy.

In a further embodiment, the present invention provides a method of haplotype phasing comprising generating a plurality of read pairs from a single DNA molecule in vitro and using the read pairs to assemble a plurality of contigs of said DNA molecule, wherein At least 1% of read pairs span a distance greater than 30 kB on the single DNA molecule and are haplotype-phased with greater than 70% accuracy. In some embodiments, at least 10% of read pairs span a distance greater than 30 kB on said single DNA molecule. In other embodiments, at least 1% of read pairs span a distance greater than 50 kB on said single DNA molecule. In other embodiments, haplotype phasing is performed with greater than 90% accuracy. In other embodiments, haplotype phasing is performed with greater than 70% accuracy.

In some embodiments, the invention provides a method of generating a first read pair from a first DNA molecule, comprising: (a) crosslinking the first DNA molecule in vitro, wherein the first DNA molecule comprises a first DNA fragment and a second DNA fragment; (b) ligating the first DNA fragment and the second DNA fragment, thereby forming a ligated DNA fragment; and (c) sequencing the ligated DNA fragment, thereby obtaining a first read right.

In some embodiments, multiple associated molecules, eg, from reconstituted chromatin, are cross-linked to the first DNA molecule. In some embodiments, association molecules include amino acids. In further embodiments, the association molecule is a peptide or protein. In certain embodiments, the first DNA molecule is crosslinked with a fixative. In some embodiments, the fixative is formaldehyde. In some embodiments, the first DNA fragment and the second DNA are generated by cleaving the first DNA molecule. In certain embodiments, the method further comprises assembling a plurality of contigs of the first DNA molecule using the first read pair. In some embodiments, each of the first DNA fragment and the second DNA fragment is attached to at least one affinity tag, and the attached DNA fragment is captured with the affinity tag.

In a further embodiment, the method further comprises: (a) providing at least a second DNA molecule with a plurality of association molecules, such as from reconstituted chromatin; (b) crosslinking the association molecules to the second DNA molecule Two DNA molecules, thereby forming a second complex in vitro; (c) cleavage of the second complex, thereby generating a third DNA fragment and a fourth DNA fragment; (d) joining the third DNA fragment to the fourth DNA fragment , thereby forming a second ligated DNA fragment; and (e) sequencing the second ligated DNA fragment, thereby obtaining a second read pair. In some embodiments, less than 40% of the DNA fragments from said DNA molecule are ligated to DNA fragments from any other DNA molecule. In a further embodiment, less than 20% of the DNA fragments from said DNA molecule are ligated to DNA fragments from any other DNA molecule.

In other embodiments, the invention provides a method of generating a first read pair from a first DNA molecule comprising a predetermined sequence comprising: (a) providing one or more DNA binding molecules to the first DNA molecule, wherein The one or more DNA binding molecules bind to the predetermined sequence; (b) externally linking a first DNA molecule in vitro, wherein the first DNA molecule comprises a first DNA segment and a second DNA segment; (c) linking the first DNA segment a DNA fragment is ligated to a second DNA fragment, thereby forming a first ligated DNA fragment; and (d) sequencing said first ligated DNA fragment, thereby obtaining a first read pair; wherein said predetermined sequence occurs The probability of being in the read pair is affected by the binding of the DNA binding molecule to the predetermined sequence.

In some embodiments, a DNA binding molecule is a nucleic acid capable of hybridizing to a predetermined sequence. In some embodiments, the nucleic acid is RNA. In other embodiments, the nucleic acid is DNA. In other embodiments, the DNA binding molecules are small molecules. In some embodiments, the small molecule binds to the predetermined sequence with a binding affinity of less than 100 μΜ. In a further embodiment, said small molecule binds to a predetermined sequence with a binding affinity of less than 1 μΜ. In a further embodiment, the DNA binding molecule is immobilized on a surface or solid support.

In some embodiments, the probability that a predetermined sequence occurs in a read pair is decreased. In other embodiments, the probability that a predetermined sequence occurs in a read pair is increased.

In other embodiments, the invention provides an in vitro library comprising a plurality of read pairs, each read pair comprising at least a first sequence element and a second sequence element, wherein the first sequence element and the second sequence element are derived from a single DNA molecules, wherein at least 1% of read pairs comprise a first sequence element and a second sequence element that are at least 50 kB apart on said single DNA molecule.

In some embodiments, at least 10% of read pairs comprise a first sequence element and a second sequence element that are at least 50 kB apart on a single DNA molecule. In other embodiments, at least 1% of read pairs comprise a first sequence element and a second sequence element that are at least 100 kB apart on a single DNA molecule.

In a further embodiment, less than 20% of read pairs comprise one or more predetermined sequences. In a further embodiment, less than 10% of read pairs comprise one or more predetermined sequences. In a further embodiment, less than 5% of read pairs comprise one or more predetermined sequences.

In some embodiments, the predetermined sequence is determined by one or more nucleic acids capable of hybridizing to the predetermined sequence. In some embodiments, the one or more nucleic acids are RNA. In other embodiments, the one or more nucleic acids are DNA. In further embodiments, the one or more nucleic acids are immobilized to a surface or solid support.

In other embodiments, the predetermined sequence is determined by one or more small molecules. In some embodiments, the one or more small molecules bind to the predetermined sequence with a binding affinity of less than 100 μΜ. In further embodiments, said one or more small molecules bind to the predetermined sequence with a binding affinity of less than 1 μΜ.

In some embodiments, the invention provides a composition comprising a DNA fragment and a plurality of associated molecules, for example, from reconstituted chromatin, wherein: (a) the associated molecules are complexed in vitro cross-linking with DNA fragments; and (b) the in vitro complex is immobilized on a solid support.

In other embodiments, the present invention provides a composition comprising a DNA fragment, a plurality of association molecules, and a DNA binding molecule, wherein: (a) said DNA binding molecule binds to a predetermined sequence of said DNA fragment; and (b) said association molecule is cross-linked to said DNA fragment.

In some embodiments, a DNA binding molecule is a nucleic acid capable of hybridizing to a predetermined sequence. In some embodiments, the nucleic acid is RNA. In other embodiments, the nucleic acid is DNA. In further embodiments, the nucleic acid is immobilized to a surface or solid support.

In other embodiments, the DNA binding molecules are small molecules. In some embodiments, the small molecule binds to the predetermined sequence with a binding affinity of less than 100 μΜ. In other embodiments, the small molecule binds to the predetermined sequence with a binding affinity of less than 1 μM.

incorporated by reference

All publications, patents, and patent applications mentioned in this specification are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety, as well as any references cited therein.

Description of drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description, which illustrates exemplary embodiments employing the principles of the invention, and to the accompanying drawings, which are as follows:

Figure 1 schematically illustrates genome assembly using high-throughput sequencing reads. The genome to be assembled is shown (top). Typically, genomes have many repetitive sequences that are difficult to assemble. Randomized, high-throughput sequence data from genomes are collected (middle) and assembled into "contigs" in unique regions of the genome (bottom). Contig assembly often ends with multiple repeats. The end result is a set of thousands of contigs, whose order and orientation relative to each other is unknown. In the figure, the contigs are numbered arbitrarily from longest to shortest.

2A-D schematically illustrate the Hi-C-based workflow of the present invention: (A) shows where DNA is cross-linked and processed to generate biotinylated ligated fragments for sequencing; Contact map data on human chromosome 14 for multiple restriction enzymes. As shown, most contacts are localized along the chromosomes.

Figure 3A-C provides the method of the present invention to assist genome assembly using Hi-C sequence data: (A) schematically shows the position of cross-linking and processing DNA using Hi-C-based workflow; (B) illustrates the alignment of reads to data to the position of assembled contigs generated from random shotgun sequencing and assembly; and (C) illustrates the construction of a contiguity summarizing read pair data between all contigs after filtering and weighting matrix. The matrix can be rearranged to show the correct organizational path. As shown, most read pairs will map within the contig. From this, it is possible to know the distribution of contact distances (see, for example, FIG. 6 ). Read pairs that map to different contigs provide data on which contigs are adjacent in a correct genome assembly.

Figure 4 shows an exemplary process flow of the present invention: first generate and prepare DNA fragments; then carry out chromatin assembly and biotinylation in vitro; then fix the chromatin/DNA complex with formaldehyde and fix it with streptavidin (streptavadin ) beads; the complex is then restriction digested to generate cohesive ends, which are then filled in with biotinylated dCTP and internal, sulfated GTP; after blunt end ligation, chromatin/DNA The complex is subjected to protease digestion, exonuclease digestion, and shearing; the DNA fragments are then pulled down with biotin and ligated with sequencing adapters; finally, the DNA fragments are selected by size and sequenced.

Figures 5A-B illustrate ambiguities in genome assembly and alignment arising from repetitive regions in the genome. (A) Uncertainty in linkage due to read pairs not being able to span duplicate regions. (B) Fragment alignment is indeterminate because read pairs cannot span marginal repeats.

Figure 6 illustrates the distribution of genomic distances between read pairs from a human XLRP library. The maximum distance achievable with other techniques is indicated for comparison.

Figure 7 illustrates the phasing accuracy for sample NA12878 of a well-characterized haplotype. Distances shown are between phased SNPs.

Figure 8 illustrates various components of an exemplary computer system according to various embodiments of the invention.

Figure 9 is a block diagram illustrating the architecture of an exemplary computer system that may be employed in connection with various embodiments of the present invention.

Figure 10 is a diagram illustrating an exemplary computer network that may be employed in connection with various embodiments of the present invention.

Figure 11 is a block diagram illustrating the architecture of another exemplary computer system that may be employed in connection with various embodiments of the present invention.

Detailed ways

As used herein and in the appended claims, the singular forms "a/an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a contig" includes a plurality of such contigs, and reference to "detecting the physical layout of a chromosome" includes reference to one or more methods for detecting the physical layout of a chromosome and those known to those skilled in the art. known equivalents, and so on.

Also, the use of "and" means "and/or" unless stated otherwise. Likewise, "comprises," "including," "containing," and "having" are interchangeable and are not intended to be limiting.

It should be further understood that when the term "comprising" is used in the description of different embodiments, those skilled in the art will understand that in some specific examples, the embodiment can use the language "consisting essentially of" or "consisting of" instead descriptively.

As used herein, the term "sequenced read" means a DNA fragment in which the sequence has been determined.

The term "contig" as used herein means a contiguous region of a DNA sequence. "Contigs" can be determined by a variety of methods known in the art, such as by aligning the sequencing reads against overlapping sequences, and/or by aligning the sequencing reads to a database of known sequences to identify which sequencing reads A segment has a high probability of being contiguous.

The term "subject" as used herein may mean any eukaryotic or prokaryotic organism.

As used herein, the term "naked DNA" may mean DNA that is substantially free of complex proteins. For example, it can mean DNA complexed with less than about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, or about 1% of endogenous proteins found in the nucleus.

The term "reconstituted chromatin" as used herein may mean chromatin formed by complexing isolated nucleoproteins with naked DNA.

As used herein, the term "read pair" may mean two or more elements that are associated to provide sequence information. In some cases, the number of read pairs may mean the number of mappable read pairs. In other cases, the number of read pairs may mean the total number of read pairs generated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the methods and compositions of the present invention, exemplary methods and materials are now described.

The present invention provides a method for generating very long range read pairs and utilizing this data for enhancing all of the above purposes. In some embodiments, the present invention provides methods for generating highly contiguous and accurate assemblies of the human genome using only about 300 million read pairs. In other embodiments, the present invention provides methods for phasing 90% or more of heterozygous variants in the human genome with an accuracy of 99% or more. Furthermore, the range of read pairs generated by the present invention can be extended across greater genomic distances. The assembly was generated from a standard shotgun library in addition to the very long read pair library. In other embodiments, the invention provides software capable of utilizing both sets of sequencing data. A single long-range read is used to generate phased variants from a library from which reads are mapped to a reference genome and subsequently used to assign variants to one of the individual's two parental chromosomes. Finally, the present invention allows extraction of larger DNA fragments using known techniques to generate exceptionally long reads.

The mechanism by which these repeats impede the assembly and alignment process is rather simple and ultimately obscures the results (Fig. 5). In the case of large repeat regions, the difficulty is spanning. Regions bordering repetitive elements cannot be confidently joined if the reads or read pairs are not long enough to span the repetitive region. In the case of smaller repeating elements, the problem is mainly layout. When a region is flanked by two repetitive elements (which is very common in genomes), it is also difficult, if not impossible, to determine its exact arrangement because the flanking elements are similar to other elements of its class. In both cases, the lack of discriminative information among repeat elements makes identification and assignment of specific repeat elements challenging. There is a need to be able to experimentally establish connections between unique fragments surrounded or separated by repeating regions.

The method of the present invention greatly advances the field of genomics by overcoming the formidable hindrance posed by these repetitive regions, thereby enabling significant advances in multiple areas of genomic analysis. To accomplish de novo assembly with previous techniques, one must either accept fragmentation of the assembly into multiple small scaffolds, or spend significant time and resources generating large insert libraries, or use other methods to generate more contiguous assemblies. These methods may include obtaining very deep sequencing coverage, constructing BAC or fosmid libraries, optical mapping or, most likely, some combination of these methods with other techniques. High resource and time requirements make these methods difficult to adopt in most small laboratories and limit the study of non-model organisms. Because the methods described here are capable of generating very long-range read pairs, de novo assembly can be accomplished with a single round of sequencing. This would reduce assembly costs by orders of magnitude and reduce the time required from months or years to weeks. In some cases, the methods disclosed herein result in less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, Multiple read pairs are generated within a range of days, less than 6 days, less than 5 days, less than 4 days, or between any two of the above specified time periods. For example, the method allows multiple read pairs to be generated in about 10 to 14 days. Even for organisms of the smallest habitats, constructing biological groups will become routine, phylogenetic analysis will not face lack of comparisons, and projects such as Genome 10k will be realized.

Likewise, structural and phasing analysis for medical purposes remains challenging. There is alarming heterogeneity in cancer, between individuals with the same type of cancer, or even within the same tumor. Eliminating the causes of the resulting effects requires a high degree of accuracy and throughput at a low cost per sample. In the field of personalized medicine, one of the gold standards for genomic medicine is a fully characterized and phased sequenced genome of all variants, including large and small structural rearrangements and novel mutations. To achieve this with previous techniques requires work similar to that required for de novo assembly, which is currently still too expensive and laborious to be a routine medical procedure. The disclosed method enables the rapid and low-cost generation of complete, accurate genomes, which could play a highly sought-after role in the study and treatment of human disease.

Finally, applying the methods disclosed herein to phasing can combine the convenience of statistical methods with the accuracy of family analysis, saving money, manpower and samples compared to using other methods alone. De novo variant phasing, a highly desirable phasing assay that has been prohibitive with previous techniques, can be readily accomplished with the methods disclosed herein. This is especially important given that the vast majority of human variants are rare (less than 5% minor allele frequency). Phasing information is very valuable for population genetic studies, which derive significant advantages from networks of highly connected haplotypes (collections of variants assigned to individual chromosomes) over segregated genotypes. Haplotype information enables higher resolution studies of historical changes in population size, migration, and exchange between subpopulations, and allows us to trace specific variants back to parents and grandparents. This in turn suggests the genetic transmission of disease-associated variants and, when multiple variants arise in a single individual, interactions between variants. The methods of the present invention may ultimately enable the preparation, sequencing and analysis of extremely long-range read pair (XLRP) libraries.

In some embodiments of the invention, a tissue or DNA sample from a subject can be provided and the method can return an assembled genome, alignment to identified variants (including large structural variants), phasing variant calling (variant call) or any additional analysis. In other embodiments, the methods disclosed herein provide an XLRP library directly to an individual.

In various embodiments of the invention, the methods disclosed herein can generate extremely long-range read pairs separated by large distances. The upper limit of this distance can be increased with the ability to collect large size DNA samples. In some cases, read pairs can span up to 50kbp, 60kbp, 70kbp, 80kbp, 90kbp, 100kbp, 125kbp, 150kbp, 175kbp, 200kbp, 225kbp, 250kbp, 300kbp, 400kbp, 500kbp, 600kbp, 700kbp, 0kbp, 90 , 1500kbp, 2000kbp, 2500kbp, 3000kbp, 4000kbp, 5000kbp or greater genomic distance. In some embodiments, read pairs can span genomic distances of up to 500 kbp. In other embodiments, read pairs may span genomic distances of up to 2000 kbp. The methods disclosed herein can be integrated and built upon standard techniques in molecular biology and further adapted to improve efficiency, specificity and genome coverage. In some cases, read pairs can be less than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 or 90 day generation. In some embodiments, read pairs can be generated in less than about 14 days. In further embodiments, read pairs can be generated in less than about 10 days. In some cases, the methods of the invention can provide more than about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% of read pairs that have at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% accuracy. For example, the method can provide at least about 90% to 100% accuracy in correctly ordering and/or orienting multiple contigs.

In other embodiments, the methods disclosed herein can be used in conjunction with currently employed sequencing technologies. For example, the method can be used in combination with well-tested and/or widely adopted sequencing instruments. In further embodiments, the methods disclosed herein can be used in conjunction with techniques and methods derived from currently employed sequencing technologies.

The method of the present invention greatly simplifies de novo genome assembly of a large number of organisms. Using previous techniques, these assemblies are currently limited to short inserts from low-cost mate-pair libraries. While it is possible to generate read pairs with genomic distances of up to 40-50 kbp available using F cohesion, these are costly, unwieldy, and too short to span the longest repeat regions, including those located in In the centromere, the size is 300kbp to 5Mbp in humans. The methods disclosed herein can provide read pairs capable of spanning large distances (eg, megabases or longer), thereby overcoming these backbone integrity issues. Thus, by utilizing the methods of the present invention, generating chromosome-level assemblies can become routine. More laborious approaches to assembly—which currently cost research laboratories an incredible amount of time and money and fail to generate large-scale genome catalogs—could become unnecessary, saving resources for more meaningful analyses. Likewise, the availability of long-range phasing information can greatly additionally assist population genomic studies, phylogenetic studies, and disease studies. The methods disclosed herein enable accurate phasing of large numbers of individuals, extending the breadth and depth of our ability to probe genomes at the population and deep-time levels.

In the field of personalized medicine, XLRP read pairs generated by the methods disclosed herein represent a meaningful advance in the accurate, low-cost, phased, and rapid generation of individual genomes. Existing methods are not capable of phasing variants over long distances, hampering the characterization of the phenotypic effects of compound heterozygous genotypes. Furthermore, structural variants with substantial impact on genomic disease are difficult to accurately identify and characterize with current technologies due to their large size compared to the reads and read-pair inserts used to study them. Read pairs spanning tens of thousands of bases to millions of bases or longer can help address this difficulty, enabling highly parallel and individualized analysis of structural variants.

Advancing basic evolutionary and biomedical research through technological advances in high-throughput sequencing. While whole genome sequencing and assembly used to be the source of large genome sequencing centers, commercially available sequencers are now low enough in cost that most research universities own one or more of these machines. Generating large amounts of DNA sequence data is currently relatively cheap. However, it is theoretically and practically difficult to generate high-quality, highly contiguous genome sequences with existing technologies. Furthermore, because most organisms of interest for analysis are diploid, including humans, each individual has two haploid copies of the genome. At heterozygous loci (eg, where the alleles from the mother differ from the alleles from the father), it is difficult to know which set of alleles came from which parent (known as haploid phasing). This information can be used to conduct a variety of evolutionary and biomedical studies, such as disease and trait association studies.

In various embodiments, the present invention provides methods for genome assembly that combine DNA preparation techniques and paired-end sequencing for high-throughput discovery of short, medium, and long junctions in a given genome. The invention further provides methods of using these linkages to facilitate genome assembly for haplotype phasing and/or metagenomic studies. While the methods provided herein can be used to determine the assembly of a subject's genome, it should also be understood that the methods provided herein can also be used to determine the assembly of parts of a subject's genome, such as chromosomes, or the composition of subject chromatin of varying lengths. Assemble.

In some embodiments, the invention provides one or more methods disclosed herein comprising a method of generating a plurality of contigs from sequenced fragments of target DNA obtained from a subject. Long stretches of target DNA can be fragmented by cutting the DNA with one or more restriction enzymes, shearing the DNA, or a combination of both. The resulting fragments are sequenced using high-throughput sequencing methods to obtain multiple sequencing reads. Examples of high-throughput sequencing methods that can be used in conjunction with the methods of the invention include, but are not limited to, 454 pyrosequencing developed by Roche Diagnostics, "clusters" sequencing developed by Illumina, SOLiD developed by Life Technologies and ion semiconductor sequencing, and DNA nanosphere sequencing developed by Complete Genomics. The overlapping ends of the different sequencing reads can then be assembled to form contigs. Alternatively, fragmented target DNA can be cloned into a vector. Cells or organisms are then transfected with the DNA vector to form a library. After replicating the transfected cells or organisms, the vectors are isolated and sequenced to generate multiple sequencing reads. The overlapping ends of the different sequencing reads can then be assembled to form contigs.

As shown in Figure 1, genome assembly, especially using high-throughput sequencing technologies, can be problematic. Assemblies often consist of thousands or tens of thousands of short contigs. The order and orientation of these contigs are often unknown, limiting the usefulness of genome assemblies. Techniques exist to sequence and orient these backbones, but they are often expensive, labor-intensive, and often fail to discover very long-range interactions.

Samples containing target DNA for contig generation can be obtained from a subject in a number of ways, including by extraction of bodily fluids such as blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat, and semen), tissue extraction, or by harvesting cells/organisms. The sample obtained may include a single type of cell/organism or may include multiple types of cells/organisms. DNA can be extracted and prepared from a sample from a subject. For example, a sample can be treated to lyse polynucleotide-containing cells using known lysis buffers, sonication techniques, electroporation, and the like. Target DNA can be further purified to remove contaminants, such as proteins, by using ethanol extraction, cesium gradients, and/or column chromatography.

In other embodiments of the invention, a method of extracting very high molecular weight DNA is provided. In some cases, data from XLRP libraries can be improved by increasing the fragment size of the input DNA. In some embodiments, extracting megabase-sized DNA fragments from cells can generate read pairs in the genome that are separated by millions of bases. In some cases, the read pairs generated can provide sequence information spanning greater than about 10 kB, about 50 kB, about 100 kB, about 200 kB, about 500 kB, about 1 Mb, about 2 Mb, about 5 Mb, about 10 Mb, or about 100 Mb. In some embodiments, a read pair can provide sequence information with a span greater than about 500 kB. In further embodiments, read pairs may provide sequence information with a span greater than about 2 Mb. In some cases, very gentle cell lysis (Teague, B. et al., (2010) Proc. Nat. Acad. Sci. USA107 (24), 10848–53) and agarose block embedding (Schwartz, D.C. and Cantor, C.R. (1984) Cell, 37(1), 67-75) extracted this very high molecular weight DNA. In other cases, commercially available machines capable of purifying DNA molecules to millions of bases in length can be used to extract very high molecular weight DNA.

In various embodiments, the present invention provides one or more of the methods disclosed herein comprising the step of probing the physical layout of chromosomes in living cells. Examples of techniques for probing the physical layout of chromosomes by sequencing include "C" family technologies such as chromosome conformation capture ("3C"), circularized chromosome conformation capture ("4C"), carbon copy chromosome capture (carbon-copy chromosome capture, "5C") and Hi-C-based methods; and chromatin immunoprecipitation (ChIP)-based methods, such as chromatin immunoprecipitation-loop (ChIP-loop), chromatin immunoprecipitation Precipitation - paired end tagging (ChIP-PET). These techniques exploit the fixation of chromatin in living cells to enhance spatial relationships within the nucleus. Subsequent processing and sequencing of the products allows the investigator to obtain a matrix of proximity associations between genomic regions. With further analysis, these associations can be used to generate a three-dimensional geometric map of the chromosomes as they are physically arranged in the nucleus of a living cell. These techniques describe the discrete spatial organization of chromosomes in living cells and enable the precise visualization of functional interactions between chromosomal loci. A problem that plagues these functional studies is the presence of nonspecific interactions, which appear in the data only due to associations of chromosomal proximity. In the present invention, these non-specific intrachromosomal interactions are captured by the methods presented herein to provide valuable information for assembly.

In some embodiments, intrachromosomal interactions are associated with chromosomal connectivity. In some cases, intrachromosomal data can assist in genome assembly. In some cases, chromatin was reconstituted in vitro. This may be advantageous because in 3C, 4C, 5C, and Hi-C, the most commonly used "C" family technologies for detection of chromatin conformation and structure by sequencing, chromatin—particularly its major protein Proteins - important for immobilization.

Figure 2 summarizes the chromatin conformation capture technique. Briefly, crosslinks are created between physically closely adjacent genomic regions. Cross-linking of proteins (eg, histones) with DNA molecules, eg, genomic DNA, within chromatin can be accomplished according to suitable methods as further detailed elsewhere herein or known in the art. In some cases, two or more nucleotide sequences can be cross-linked by a protein that binds to one or more nucleotide sequences. One approach is to expose chromatin to ultraviolet radiation (Gilmour et al., Proc. Nat'l. Acad. Sci. USA 81:4275-4279, 1984). Polynucleotide fragments may also be crosslinked using other methods, such as chemical or physical (eg optical) crosslinking. Suitable chemical cross-linking agents include, but are not limited to, formaldehyde and psoralen (Solomon et al., Proc. Nat L. Acad. Sci. USA 82:6470-6474, 1985; Solomon et al., Cell 53:937-947, 1988). For example, crosslinking can be performed by adding 2% formaldehyde to a mixture containing DNA molecules and chromatin proteins. Examples of other reagents that can be used to cross-link DNA include, but are not limited to, UV light, mitomycin C, nitrogen mustard, melphalan, 1,3-butadiene diepoxide, cis-diamine dichloroplatinum(II), and cyclic phosphoramide. Suitably, the cross-linking agent will form bridges over relatively short distances - for example about - cross-linking, thereby selecting tight interactions that can be reversed.

In some embodiments, DNA molecules can be co-immunoprecipitated either before or after crosslinking. In some cases, DNA molecules can be fragmented. The fragments can be contacted with a binding partner, such as an antibody that specifically recognizes and binds to an acetylated histone, such as H3. Examples of such antibodies include, but are not limited to, acetylated histone H3 antibodies, which are available from Upstate Biotechnology (Lake Placid, N.Y.). Polynucleotides from the immunoprecipitate can then be harvested from the immunoprecipitate. Acetylated histones can cross-link to adjacent polynucleotide sequences prior to fragmenting chromatin. The mixture is then processed to separate the polynucleotides in the mixture. Separation techniques are known in the art and include, for example, shearing techniques to generate smaller genomic fragments. Fragmentation can be performed using established methods for fragmenting chromatin, including, for example, sonication, shearing, and/or use of restriction enzymes. Restriction enzymes can have restriction sites that are 1, 2, 3, 4, 5 or 6 bases in length. Examples of restriction enzymes include, but are not limited to, AatII, Acc65I, AccI, AciI, Acll, Acul, AfeI, AflII, AflIII, AgeI, AhdI, AleI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI , AseI, AsiSI, AvaI, AvaII, AvrII, BaeGI, BaeI, BamHI, BanI, BanII, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BfaI, BfuAI, BfuCI, BglI, BglII, BlpI, BmgBI , BmrI, BmtI, BpmI, BpulOI, BpuEI, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaWI, BsaXI, BscRI, BscYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI, BsmFI, BsmI, BsoBI, Bsp1286I , BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFI, BsrGI, BsrI, BssHII, BssKI, BssSI, BstAPI, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, BstZ17I, Bsu36I, BtgI, BtgZI , BtsCI, BtsI, Cac8I, ClaI, CspCI, CviAII, CviKI-1, CviQI, DdcI, DpnI, DpnII, DraI, DraIII, DrdI, EacI, EagI, EarI, EciI, Eco53kI, EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRV , FatI, FauI, Fnu4HI, FokI, FseI, FspI, HaeII, HaeIII, HgaI, HhaI, HincII, HindIII, HinfI, HinPlI, HpaI, HpaII, HphI, Hpy166II, Hpy188I, Hpy188III, Hpy99I, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V , KasI, KpnI, MboI, MboII, MfeI, MluI, MlyI, MmeI, MnlI, MscI, MseI, MslI, MspAlI, MspI, MwoI, NaeI, NarI, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI , N ciI, NcoI, NdeI, NgoMIV, NheI, NlaIII, NlaIV, NmeAIII, NotI, NruI, NsiI, NspI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, PacI, PaeR7I, PciI, PflFI, PflMI, PhoI, PleI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspGI, PspOMI, PspXI, PstI, PvuI, PvuII, RsaI, RsrII, SacI, SacII, SalI, SapI, Sau3AI, Sau96I, SbfI, ScaI, ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SspI, StuI, StyD4I, StyI, SwaI, T, TaqαI, TfiI, TliI, TseI, Tsp45I, Tsp509I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XmaI, XmnI, and ZraI. The resulting fragments can vary in size. The resulting fragments may also contain single-stranded overhangs at the 5' or 3' ends.

In some embodiments, fragments of about 100 to 5000 nucleotides can be obtained using sonication techniques. Alternatively, fragments of about 100 to 1000, about 150 to 1000, about 150 to 500, about 200 to 500, or about 200 to 400 nucleotides may be obtained. Samples can be prepared as cross-linked concatenated read fragments for sequencing. In some cases, single, short stretches of polynucleotides can be produced, for example, by joining two sequence fragments that cross-link intramolecularly. Sequence information can be obtained from a sample using any suitable sequencing technique described in further detail herein or known in the art, eg, high-throughput sequencing. For example, ligation products can be paired-end sequenced to obtain sequence information from each end of the fragment. Pairings of sequence segments can be described in the resulting sequence information, which is associated with haplotype information along the linear distance separating the two sequence segments along the polynucleotide.

One of the characteristics of the data generated by Hi-C is that most read pairs are found to be in close linear proximity when mapped back to the genome. That is, most read pairs are found to be close to each other in the genome. In the resulting data set, the probability of intrachromosomal contacts was on average much higher than that of interchromosomal contacts, as expected if chromosomes occupy different regions. Furthermore, loci on the same chromosome separated by even >200 Mb are more likely to interact than loci on different chromosomes, although the probability of interaction decreases dramatically with linear distance. In the detection of long distance intrachromosomal and especially interchromosomal contacts, the "background" of the short and intermediate distance intrachromosomal contacts is the background noise to be removed using Hi-C analysis.

Remarkably, Hi-C assays in eukaryotes have shown that, in addition to species-specific and cell-type-specific chromatin interactions, there are two canonical types of interactions. One type is distance-dependent decay (DDD), the general trend of decay in interaction frequency as a function of genomic distance. The second type of cis-trans ratio (cis-trans ratio, CTR) is a significantly higher interaction frequency between loci located on the same chromosome than between loci on different chromosomes, even when genes on the same chromosome Blocks are separated by tens of millions of bases. These patterns may reflect overall polymer dynamics (where proximal loci have a higher probability of random interactions), as well as specific nuclear organization features such as the formation of chromosomal regions, the tendency of interphase chromosomes to occupy A phenomenon in which different spaces within the nucleus hardly mix. Although the exact details of these two types may vary between species, cell types, and cell conditions, they are ubiquitous and prominent. These types are so strong and consistent that they are used to assess trial quality and are often normalized across data to reveal detailed interactions. However, in the methods disclosed herein, genome assembly can take advantage of the three-dimensional structure of the genome. The features that make canonical Hi-C interaction types a hindrance to the analysis of specific loop interactions, namely their prevalence, strength, and consistency, can be used as powerful tools for estimating the genomic position of contigs.

In particular embodiments, examining the physical distance between read pairs within a chromosome reveals some useful features of genome assembly-related data. First, shorter-range interactions are more common than longer-range interactions (see, for example, Figure 6). In other words, each read in a read pair is more likely to be matched with a region that is close in the actual genome than a region that is far away. Second, medium- and long-range interactions have long tails. In other words, the read pairs carry information about the intrachromosomal alignment over kilobase (kB) or even million base (Mb) distances. For example, a read pair can provide sequence information that spans greater than about 10 kB, about 50 kB, about 100 kB, about 200 kB, about 500 kB, about 1 Mb, about 2 Mb, about 5 Mb, about 10 Mb, or about 100 Mb. These data characteristics simply indicate that adjacent genomic regions on the same chromosome are more likely to be in close physical proximity—an expected outcome, since they are chemically linked to each other by the DNA backbone. Presumably genome-wide chromatin interaction datasets, such as those generated by Hi-C, would provide long-range information about the grouping and linear organization of sequences along entire chromosomes.

Despite the simplicity and relatively low cost of assays for Hi-C, existing protocols for genome assembly and haplotype analysis require 10 ⁶ –10 ⁸ cells, which is not possible with considerable amounts of material, especially From certain human patient samples. In contrast, the methods disclosed herein include methods that allow accurate and predictive results for genotype assembly, haplotype phasing, and metagenomics with significantly less cell-derived material. For example, less than about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1.0 μg can be used using the methods disclosed herein. μg, about 1.2μg, about 1.4μg, about 1.6μg, about 1.8μg, about 2.0μg, about 2.5μg, about 3.0μg, about 3.5μg, about 4.0μg, about 4.5μg, about 5.0μg, about 6.0μg, About 7.0 μg, about 8.0 μg, about 9.0 μg, about 10 μg, about 15 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 150 μg, about 200 μg , about 300 μg, about 400 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900 μg, or about 1000 μg of DNA. In some embodiments, the DNA used in the methods disclosed herein can be extracted from less than about 1,000,000, about 500,000, about 100,000, about 50,000, about 10,000, about 5,000, about 1,000, about 5,000 or about 1,000, about 500 or about 100 cells.

In general, methods for the physical layout of chromosomes, such as Hi-C-based techniques, utilize chromatin formed in cells/organisms, such as chromatin isolated from cultured cells or primary tissues. The present invention not only allows the use of these techniques with chromatin isolated from cells/organisms, but also remodels chromatin. Reconstituted chromatin differs from chromatin formed in cells/organisms in several features. First, for many samples, collection of naked DNA samples can be accomplished using a variety of non-invasive to invasive methods, such as by collection of bodily fluids, swabs of the oral or rectal area, epithelial samples, etc. Second, remodeling chromatin substantially prevents the formation of interchromosomal and other long-distance interactions that generate artifacts of genome assembly and haplotype phasing. In some cases, according to the methods and compositions of the present invention, a sample may have less than about 20, 15, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.4, 0.3, 0.2, 0.1% or less interchromosomal or intermolecular crosslinks. In some embodiments, a sample may have less than about 5% interchromosomal or intermolecular crosslinks. In some embodiments, a sample may have less than about 3% interchromosomal or intermolecular crosslinks. In further embodiments, the sample may have less than about 1% interchromosomal or intermolecular crosslinks. Third, the frequency of sites capable of crosslinking, and thus the frequency of intramolecular crosslinking within a polynucleotide, can be adjusted. For example, the ratio of DNA to histone can be varied so that the nucleosome density can be adjusted to a desired value. In some instances, nucleosome density is reduced below physiological levels. Accordingly, the distribution of crosslinks can be altered to favor long-range interactions. In some embodiments, subsamples with different crosslink densities can be prepared to cover both short-range and long-range associations. For example, the crosslinking conditions can be adjusted so that at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% , about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% of the crosslinking occurs between DNA fragments that are at The sample DNA is molecularly separated by at least about 50 kb, about 60 kb, about 70 kb, about 80 kb, about 90 kb, about 100 kb, about 110 kb, about 120 kb, about 130 kb, about 140 kb, about 150 kb, about 160 kb, about 180 kb, about 200 kb, about 250 kb , about 300 kb, about 350 kb, about 400 kb, about 450 kb, or about 500 kb.

In various embodiments, the present invention provides methods that enable multiple read pairs to be mapped to multiple contigs. There are several publicly available computer programs for mapping reads to contig sequences. These read mapper data also provide data describing the degree to which particular read maps within a genome are unique. From the population of reads that uniquely map into the contig with high confidence, we can infer the distribution of distances between reads in each read pair. These are the data shown in Figure 6. For those read pairs whose reads confidently map to different contigs, the mapping data imply a connection between the two contigs in question. This also implies that the distance between two contigs is proportional to the distribution of distances obtained from the above analysis. Thus, each read pair whose reads map to a different contig implies a junction between the two contigs in the correct assembly. The connections inferred from all these mapped read pairs can be summarized in an adjacency matrix, with rows and columns representing each contig. Read pairs that join contigs are marked with a non-zero value in the corresponding row and column, indicating the contig to which the reads in the read pair map. Most read pairs will map within a contig, from which a distribution of distances between read pairs can be derived, and from which an adjacency matrix for the contig can be constructed using read pairs that map to different contigs.

In various embodiments, the invention provides methods comprising constructing an adjacency matrix of contigs using read mapping data from read pair data. In some embodiments, the adjacency matrix uses a weighting scheme for read pairs that captures the tendency for short-range interactions over long-range interactions (see, eg, FIG. 3 ). Read pairs spanning shorter distances are generally more common than read pairs spanning longer distances. Using the read pair data mapped to a single contig, a function describing the probability of a particular distance can be fitted to understand this distribution. Therefore, one of the important characteristics of a read pair that maps to a different contig is its location on the contig. For read pairs that both map to one end of a contig, the inferred distance between these contigs can be short and thus the distance between contiguous reads small. Since shorter distances between read pairs are more common than longer distances, this configuration provides stronger evidence that the two contigs are adjacent than reads positioned farther from the edges of the contigs. Thus, connections in the adjacency matrix are further weighted by the distance of the reads from the contig edge. In a further embodiment, the adjacency matrix can be further rescaled to reduce the weight of a large number of contacts on some contigs that represent promiscuous regions of the genome. These regions of the genome can be identified by having a high proportion of reads mapping to them, a priori that are more likely to contain spurious reads mapping that could provide misinformation for assembly. In a further embodiment, this regulation can be directed by finding one or more conserved binding sites for one or more mediators that regulate chromatin backbone interactions, such as the transcriptional repressor CTCF, endocrine receptors, cohesin or Covalently modified histones.

In some embodiments, the invention provides one or more of the methods disclosed herein, comprising the step of analyzing an adjacency matrix to determine a path through a contig representing its sequence and/or orientation to the genome. In other embodiments, the path through the contigs can be chosen such that each contig is visited exactly once. In a further embodiment, the path through the contig is selected such that the path through the adjacency matrix maximizes the sum of edge weights visited. In this way, the most likely contig joins are proposed for correct assembly. In a further embodiment, the path through the contigs is chosen such that each contig is visited exactly once and the edge weights of the adjacency matrix are maximized.

In diploid genomes, it is often important to know which allelic variants are linked on the same chromosome. This is called haplotype phasing. Short reads from high-throughput sequence data rarely allow one to directly observe which allelic variants are linked. Computational inference of haplotype phasing is unreliable over long distances. The invention provides one or more methods that use the allelic variants on a read pair to determine which allelic variants are linked.

In various embodiments, the methods and compositions of the invention enable haplotype phasing of diploid or polyploid genomes associated with multiple allelic variants. The methods described herein can thus determine that linked allelic variants are linked based on variant information from read pairs and/or assembled contigs using the read pairs. Examples of allelic variants include, but are not limited to, those known from 1000genomes, UK10K, HapMap, or other projects for discovering genetic variation in humans. By having validated haplotype phasing data, disease associations to specific genes can be more easily unmasked, for example by the discovery of nonjoint, inactivating mutations in both copies of SH3TC2 that cause Charcot-Marie-Tooth disease (Charcot-Marie-Tooth disease). Marie-Tooth) neuropathy (Lupski JR, Reid JG, Gonzaga-Jauregui C et al., N. Engl. J. Med. 362:1181–91, 2010), and by the discovery of non-connected, Inactivating mutations, leading to hypercholesterolemia 9 (Rios J, Stein E, Shendure J et al., Hum. Mol. Genet. 19:4313–18, 2010).

Humans are heterozygous for an average of 1 locus in 1,000 loci. In some cases, a single lane of data using a high-throughput sequencing method is capable of generating at least about 150,000,000 read pairs. A read pair can be about 100 base pairs in length. From these parameters, an estimated one-tenth of all reads from human samples covered heterozygous sites. Therefore, it was estimated that on average one percent of all read pairs from human samples covered heterozygous sites. Correspondingly, approximately 1,500,000 read pairs (1 percent of 150,000,000) provided phased data using a single lane. There are about 3 billion bases in the human genome, one in a thousand is heterozygous, and the average human genome has about 3 million heterozygous sites. The number of read pairs representing a pair of heterozygous loci is about 1,500,000, and using a typical high-throughput sequencing machine, using a single lane phased by a high-throughput sequencing method, the average coverage per heterozygous locus is about (1X) . Thus, the diploid human genome can be reliably and completely phased with one lane of high-throughput sequencing data associated with sequence variants from samples prepared using the methods disclosed herein. In some embodiments, a lane of data can be a set of DNA sequence read data. In a further embodiment, the data for one lane may be a set of DNA sequence read data from a single run of a high throughput sequencing instrument.

Since the human genome consists of two homologous sets of chromosomes, understanding the true genetic makeup of an individual requires describing the maternal and paternal copies or haplotypes of the genetic material. The haplotypes obtained in an individual are useful in several ways. First, haplotypes are clinically useful in predicting donor-recipient matching outcomes in organ transplantation and are increasingly used as a way to detect disease associations. Second, in genes exhibiting compound heterozygosity, haplotypes provide information on whether two deleterious variants are located on the same allele, greatly affecting the prediction of whether inheritance of these variants is deleterious. Third, haplotypes from multiple groups of individuals have provided information on population structure and the evolutionary history of humans. Finally, the recently described pervasive allelic imbalance in gene expression suggests that genetic or epigenetic differences between alleles may contribute to quantitative differences in expression. Understanding haplotype structure will describe the mechanisms of variants that contribute to allelic imbalance.

In certain embodiments, the methods disclosed herein include in vitro techniques for immobilizing and capturing correlations between distant regions of the genome, as required for long-range ligation and phasing. In some cases, the method includes constructing and sequencing an XLRP library to yield genomically very distant read pairs. In some cases, interactions initially arise from random pairings within a single DNA segment. In some embodiments, genomic distances between fragments can be inferred because fragments that are close to each other in a DNA molecule interact more frequently with higher probability, while distant parts of the molecule will interact less frequently. Thus, there is a systematic correlation between the logarithm connecting two loci and their contiguity on the input DNA. As shown in Figure 2, the present invention can generate read pairs that span the largest DNA fragments in the extraction. The maximum length of input DNA used for this library was 150kbp, which is the longest meaningful read pair we have observed from the sequencing data. This means that if the input DNA fragments are larger, the invention can also join more distant loci on the genome. Complete genome assemblies can be achieved through the application of improved assembly software tools that are particularly suited to handle the type of data generated by the present method.

Very high phasing accuracy can be obtained by using the data generated by the methods and compositions of the present invention. Compared with previous methods, the method described here can phase a higher proportion of variants. Phasing can be achieved while maintaining a high level of accuracy. The phase information can be extended to a longer range, such as greater than about 200kbp, about 300kbp, about 400kbp, about 500kbp, about 600kbp, about 700kbp, about 800kbp, about 900kbp, about 1Mbp, about 2Mbp, about 3Mbp, about 4Mbp, about 5Mbps or about 10Mbps. In some embodiments, greater than 90% of reads for human samples can be phased with greater than 99% accuracy using fewer than about 250 million reads or read pairs, e.g., by using data from only 1 lane of Illumina HiSeq. Heterozygous SNP. In other cases, using less than about 250 million or 500 million reads or read pairs, e.g., by using data from only 1 or 2 lanes of Illumina HiSeq, results in greater than about 70%, 80%, 90%, 95% or 99% accuracy phases greater than about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of heterozygous SNPs for human samples. For example, greater than 95% or 99% of heterozygous SNPs for human samples can be phased with greater than 95% or 99% accuracy using less than about 250 million or 500 million reads or read pairs. In further cases, the length of the read can be increased to about 200bp, 250bp, 300bp, 350bp, 400bp, 450bp, 500bp, 600bp, 800bp, 1000bp, 1500bp, 2kbp, 3kbp, 4kbp, 5kbp, 10kbp, 20kbp, 50kbp or 100kbp to capture additional variants.

In other embodiments of the invention, data from XLRP libraries can be used to confirm the orientation ability of long-range read pairs. As shown in Figure 6, the accuracy of those results is on par with the state-of-the-art, but extends further to significantly longer distances. Existing sample preparation workflows for specific sequencing methods identify variant isoforms located within targeted restriction sites within read lengths, eg, 150 bp, for phasing. In one example, 44% of the 1,703,909 heterozygous SNPs present were phased with greater than 99% accuracy from an XLRP library constructed for NA12878, a reference sample for assembly. In some cases, with judicious choice of restriction enzymes or with a combination of different enzymes, this ratio can be extended to nearly all variant sites.

In some embodiments, the compositions and methods described herein allow for the study of metagenomes, such as those found within the human digestive tract. Accordingly, partial or complete genome sequences of some or all organisms inhabiting a given ecological environment can be studied. Examples include random sequencing of the entire gut microbes, microbes found on certain areas of the skin, and microbes living in toxic waste sites. The compositions and methods of the microbial populations in these environments can be determined using the compositions and methods described herein, as well as the interconnected biochemical aspects encoded by their respective genomes. The method of the present invention can realize metagenomic research from complex biological environments, such as those comprising more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250 , 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000 or more organisms and/or organism variants.

Using the methods and systems described herein, the high accuracy required for sequencing cancer genomes can be achieved. Inaccurate reference genomes may pose basecalling challenges when sequencing cancer genomes. Heterogeneous samples and small starting materials, such as those obtained from biopsies, pose additional challenges. Furthermore, for cancer genome sequencing, detection of large structural variants and/or loss of heterozygosity is often critical, as is the ability to distinguish somatic variants from errors in base calling.

The system and method of the present invention can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more Generate accurate long sequences in complex samples with diverse genomes. Mixed samples of normal, benign and/or neoplastic origin can be analyzed, optionally without normal controls. In some embodiments, as little as 100 ng, or even as few as hundreds of genome equivalents, of starting sample is used to generate accurate long sequences. The systems and methods described herein can detect large structural variants and rearrangements. Phased variant calls can be obtained along long sequences spanning about 1 kbp, about 2 kbp, about 5 kbp, about 10 kbp, 20 kbp, about 50 kbp, about 100 kbp, about 200 kbp, about 500 kbp, about 1 Mbp, about 2Mbp, about 5Mbp, about 10Mbp, about 20Mbp, about 50Mbp, or about 100Mbp or more nucleotides. For example, phased variant identification can be obtained along long sequences spanning about 1 Mbp or about 2 Mbp.

Haplotypes determined using the methods and systems described herein can be assigned to computing resources, such as computing resources on a network, such as a cloud system. Short variant calls can be revised, if necessary, using relevant information stored in the computing resource. Structural variants can be detected based on combined information from short variant identification and information stored in computational resources. To improve accuracy, uncertain parts of the genome can be reassembled, such as segmental repeats, regions prone to structural variation, highly variable and medically relevant MHC regions, centromere and telomeric regions, and other heterochromatin regions, including But not limited to those with repetitive regions, low sequence accuracy, high variation rate, ALU repeats, segmental repeats or any other relevant uncertain parts known in the art.

Sample types can be assigned to sequence information locally or in a networked computing resource, such as the cloud. Where the source of the information is known, for example when the source of the information is from cancer or normal tissue, that source can be assigned to the sample as part of the sample type. Other sample type examples generally include, but are not limited to, tissue type, sample collection method, presence of infection, type of infection, processing method, size of sample, and the like. Where a complete or partial comparative genome sequence is available, such as a normal genome aligned to a cancer genome, differences between the sample data and the comparative genome sequence can be determined and optionally output.

The methods of the present invention can be used for the analysis of genetic information of selected genomic regions of interest, as well as genomic regions that can interact with selected regions of interest. The amplification methods disclosed herein can be used with devices, kits, and methods known in the art for genetic analysis, such as, but not limited to, those found in US Pat. In some cases, the amplification methods of the invention can be used to amplify target nucleic acids for DNA hybridization studies to determine the presence or absence of polymorphisms. Polymorphisms, or alleles, can be associated with diseases or disorders such as genetic diseases. In other cases, polymorphisms may be associated with susceptibility to a disease or disorder, such as polymorphisms associated with addiction, degenerative and age-related disorders, cancer, and the like. For example, in other cases, polymorphisms may be associated with beneficial traits, such as enhanced coronary artery health or resistance to diseases such as HIV or malaria, or to regression of diseases such as osteoporosis, Alzheimer's or dementia disease resistance.

The compositions and methods of the invention are useful for diagnostic, prognostic, therapeutic, patient stratification, drug development, treatment selection and screening purposes. The present invention has the following advantages: Using the method of the present invention, a plurality of different target molecules can be analyzed from a single biomolecule sample at one time. This allows, for example, to perform several diagnostic tests on one sample.

The compositions and methods of the invention can be used in genomes. The method described in the present invention can quickly provide answers that meet this application. The methods and compositions described herein are useful in the process of discovering biomarkers useful for diagnosis or prognosis and as indicators of health and disease. The methods and compositions described herein are useful for screening drugs, eg, drug development, treatment selection, determining efficacy and/or identifying targets for drug development. The ability to test gene expression is very important in screening assays involving drugs because proteins are the final gene products in the body. In some embodiments, the methods and compositions described herein will measure protein and gene expression simultaneously, which will provide the most information relevant to the particular screen being done.

The compositions and methods of the invention can be used in gene expression analysis. The methods described herein discriminate between nucleotide sequences. Differences between target nucleotide sequences can be, for example, single nucleic acid base differences, nucleic acid deletions, nucleic acid insertions, or rearrangements. These sequence differences involving more than one base can also be detected. The methods of the invention enable the detection of infectious diseases, genetic diseases and cancer. This is also useful in environmental monitoring, forensics and food science. Examples of genetic analyzes that can be performed on nucleic acids include, for example, SNP detection, STR detection, RNA expression analysis, promoter methylation, gene expression, virus detection, virus subtyping, and drug resistance.

The method can be applied to the analysis of biomolecular samples obtained from or derived from a patient to determine whether a diseased cell type is present in the sample, the stage of the disease, the patient's prognosis, the patient's ability to respond to a particular treatment, or what is best for the patient. Treatment. The methods of the invention can also be applied to identify biomarkers for specific diseases.

In some embodiments, the methods described herein are used in the diagnosis of a condition. As used herein, the term "diagnosing" or a condition "diagnosing" may include predicting or diagnosing a condition, determining susceptibility to a condition, monitoring treatment for a condition, diagnosing treatment response to a condition, or response to a condition, progression of a condition, or specific treatment for a disease prognosis. For example, a blood sample can be assayed according to any of the methods described herein to determine the presence and/or amount of a marker of a disease or malignant cell type in the sample to diagnose or stage the disease or cancer.

In some embodiments, the methods and compositions described herein are used in the diagnosis and prognosis of disease conditions.

The methods described herein are particularly useful in the management of a number of immunological, proliferative and malignant diseases and disorders. Immune diseases and disorders include allergic diseases and disorders, disorders of immune function, and autoimmune diseases and conditions. Allergic diseases and disorders include, but are not limited to, allergic rhinitis, allergic conjunctivitis, allergic asthma, atopic eczema, atopic dermatitis, and food allergy. Immunodeficiency disorders include but are not limited to severe combined immunodeficiency (SCID), hypereosinophilic syndrome, chronic granulomatous disease, leukocyte adhesion deficiency type I and type II, high IgE syndrome, and congenital leukocyte granule abnormalities Syndrome (Chediak Higashi), neutropenia, neutropenia, hypoplasia, blood gammaglobulinemia, hyper-IgM syndrome, DiGeorge/soft palate-cardio-facial syndrome and interference Defects in the γ-TH1 pathway. Autoimmune and immunoregulatory disorders including, but not limited to, rheumatoid arthritis, diabetes, systemic lupus erythematosus, Graves' disease, Graves' ophthalmopathy, Crohn's disease, multiple sclerosis, psoriasis, systemic sclerosis goiter, goiter and lymphomatous goiter (Hashimoto's thyroiditis, lymphocytic goiter), alopecia areata, autoimmune myocarditis, lichen sclerosus, autoimmune uveitis, Addison's disease, atrophy gastritis, myasthenia gravis, idiopathic thrombocytopenic purpura, hemolytic anemia, primary biliary cirrhosis, Wegener's granulomatosis, polyarteritis nodosa, and inflammatory bowel disease, allogeneic Allograft rejection and tissue destruction from allergic reactions to infectious microorganisms or environmental antigens.

Proliferative diseases and disorders that may be assessed by the methods of the invention include, but are not limited to, neonatal angiomatosis; secondary progressive multiple sclerosis; chronic progressive myeloid degenerative disease; neurofibromas; keloid formation; osteitis deformans, fibrocystic disease (e.g. breast or uterine); sarcoidosis; Peronies and Duputren's fibrosis, liver cirrhosis, atherosclerosis and Vascular restenosis.

Malignant diseases and disorders that can be assessed by the methods of the invention include both hematological malignancies and solid tumors.

Hematological malignancies are particularly treatable by the methods of the invention when the sample is a blood sample, since such malignancies involve changes in blood-borne cells. Such malignancies include non-Hodgkin's lymphoma, Hodgkin's lymphoma, non-B cell lymphoma and other lymphomas, acute or chronic leukemia, polycythemia, thrombocytosis, multiple myeloma, bone marrow Dysplastic syndromes, myeloproliferative disorders, myelofibrosis, atypical immune lymphoproliferative and plasma cell abnormalities.

Plasma cell abnormalities that can be assessed by the methods of the invention include multiple myeloma, amyloidosis, and essential macroglobulinemia.

Examples of solid tumors include, but are not limited to, rectal cancer, breast cancer, lung cancer, prostate cancer, brain tumors, central nervous system tumors, bladder tumors, melanoma, liver cancer, osteosarcoma and other bone cancers, testicular and ovarian cancers, head and neck cancers tumors of the neck and cervix.

The methods of the invention can also detect genetic diseases. This can be done through prenatal or postnatal screening for chromosomal and gene aberrations or for genetic diseases. Examples of genetic disorders that can be tested include: 21-hydroxylase deficiency, cystic fibrosis, fragile X syndrome, Turner syndrome, Duchenne muscular dystrophy, Down syndrome or other trisomy, cardiac disease, monogenic disease, human leukocyte antigen (HLA) typing, phenylketonuria, sickle cell anemia, Tay-Sachs disease, thalassemia, Klinefelter syndrome, Tay-Sachs disease, Huntington's disease, Autoimmune diseases, lipidosis, obesity deficiency, hemophilia, inborn errors of metabolism and diabetes.

The methods of the present invention can be used to diagnose pathogenic infections, such as intracellular bacterial and viral infections, by determining the presence and/or amount of markers of bacteria or viruses, respectively, in a sample.

The methods of the invention can detect a variety of infectious diseases. These infections can be caused by bacterial, viral, parasitic and fungal infectious agents. Drug resistance of various infectious agents can also be determined using the invention.

Bacterial infectious agents detectable by the present invention include Escherichia coli, Salmonella, Shigella, Klebsiella, Pseudomonas, mononucleosis Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium intracellulare, Yersinia, Francisella, Pasteurella ( Pasteurella), Brucella, Clostridia, Bordetella pertussis, Bacteroides, Staphylococcus aureus, Streptococcus pneumonia ), B-Hemolytic streptococcus (B-Hemolyticstrep.), Corynebacteria (Corynebacteria), Legionella (Legionella), Mycoplasma (Mycoplasma), Ureaplasma (Ureaplasma), Chlamydia (Chlamydia), Neisseria gonorrhoeae (Neisseria gonorrhea), Neisseria meningitides, Hemophilus influenza, Enterococcus faecalis, Proteus vulgaris, Proteusmirabilis, Helicobacter pylori pylori), Treponema palladium, Borrelia burgdorferi, Borrelia recurrentis, Rickettsial pathogens, Nocardia and actin bacteria (Acitnomycetes).

Fungal infectious agents detectable by the present invention include Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Paracoccidioides brasiliensis (Paracoccidioides brasiliensis), Candida albicans, Aspergillus fumigautus, algae (Phycomycetes (Rhizopus)), Sporothrix schenckii, pigmented mycoses ( Chromomycosis) and Maduromycosis.

Viral infectious agents detectable by the present invention include human immunodeficiency virus, human T lymphocyte virus, hepatitis viruses (such as hepatitis B virus and hepatitis C virus), Epstein-Barr virus (Epstein-Barr virus), giant Cytoviruses, human papillomaviruses, orthomyxoviruses, paramyxoviruses, adenoviruses, coronaviruses, rhabdoviruses, polioviruses, togaviruses, bunya viruses, arenaviruses (arenaviruses), rubella viruses (rubella viruses) and reo viruses (reo viruses).

The parasitic medium detectable by the present invention includes Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodium ovale, Onchoverva volvulus), Leishmania, Trypanosoma spp., Schistosoma spp., Entamoebahistolytica, Cryptosporidium, Giardia spp. ), Trichimonas spp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enterobius vermicularis, roundworm ( Ascaris lumbricoides), Trichuris trichiura, Dracunculus medinsis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystis carinii carinii) and Necatoramericanis.

The invention can also be used to detect drug resistance of infectious agents. Examples include vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, multidrug-resistant Mycobacterium tuberculosis, and azidothorax-resistant Glycoside human immunodeficiency virus can be identified by the present invention.

Accordingly, target molecules detected using the compositions and methods of the invention may be markers of the patient (eg, cancer markers) or markers of infection with a foreign agent, eg, bacterial or viral markers.

The compositions and methods of the invention can be used to identify and/or quantify target molecules, the abundance of which is indicative of a biological state or disease condition, such as blood markers that are up- or down-regulated with the disease.

In some embodiments, the methods and compositions of the invention are useful for cytokine expression. The low sensitivity of the methods described herein will facilitate early detection of cytokines (eg, cytokines as biomarkers of a condition), diagnosis or prognosis of a disease such as cancer, and identification of subclinical conditions.

The different samples from which the target polynucleotides are derived can comprise multiple samples from the same individual, samples from different individuals, or a combination thereof. In some embodiments, a sample comprises multiple polynucleotides from the same individual. In some embodiments, a sample comprises a plurality of polynucleotides from two or more individuals. An individual is any organism or part of an organism from which a target polynucleotide is derived, non-limiting examples of which include plants, animals, fungi, protists, akaryotes, viruses, mitochondria, and chloroplasts. A sample polynucleotide can be isolated from a subject, eg, a cell sample, tissue sample, or organ sample derived from the subject, including, eg, a cultured cell line, a biopsy, a blood sample, or a blood sample containing cells. A subject can be an animal, including but not limited to animals such as cows, pigs, mice, rats, chickens, cats, dogs, and typically mammals, such as humans. Samples may also be obtained artificially, for example by chemical synthesis. In some embodiments, the sample comprises DNA. In some embodiments, the sample comprises genomic DNA. In some embodiments, the sample comprises mitochondrial DNA, chloroplast DNA, plasmid DNA, bacterial artificial chromosomes, yeast artificial chromosomes, oligonucleotide tags, or combinations thereof. In some embodiments, the sample includes DNA generated from a primer extension reaction using any suitable combination of primers and DNA polymerase, including but not limited to polymer chain reaction (PCR), reverse transcription, and combinations thereof . When the template used in the primer extension reaction is RNA, the reverse transcription product is called complementary DNA (cDNA). Primers useful in primer extension reactions can include sequences specific for one or more targets, random sequences, partially random sequences, and combinations thereof. Reaction conditions suitable for primer extension reactions are known in the art. In general, a sample polynucleotide includes any polynucleotide present in a sample, which may or may not contain a target polynucleotide.

In some embodiments, nucleic acid template molecules (eg, DNA or RNA) are isolated from biological samples that contain various other components, such as proteins, lipids, and non-template nucleic acids. Nucleic acid template molecules can be obtained from any cellular material obtained from animals, plants, bacteria, fungi or any other cellular organism. Biological samples for use in the present invention include viral particles or preparations. Nucleic acid template molecules can be obtained directly from the organism or from biological samples derived from the organism, such as blood, urine, cerebrospinal fluid, semen, saliva, sputum, stool, and tissue. Any tissue and body fluid sample can be used as a source of nucleic acid for use in the present invention. Nucleic acid template molecules can also be isolated from cultured cells, such as primary cell cultures or cell lines. The cell or tissue from which the template nucleic acid molecule is derived may be infected by a virus or other intracellular pathogen. A sample can also be total RNA, a cDNA library, viral or genomic DNA extracted from a biological sample. The sample can also be isolated DNA from a non-cellular source, such as amplified/isolated DNA from a freezer.

Methods for extracting and purifying nucleic acids are known in the art. For example, nucleic acids can be purified by organic extraction with phenol, phenol/chloroform/isoamyl alcohol, or similar formulations, including TRIzol and TriReagent. Other non-limiting examples of extraction techniques include: (1) ethanol precipitation followed by organic extraction such as with phenol/chloroform organic reagents (Ausubel et al., 1993), with or without automated nucleic acid extractors such as those available from Applied Biosystems (Foster City , Calif.) model 341 DNA extractor; (2) stationary phase adsorption (US Pat. No. 5,234,809; Walsh et al., 1991); and (3) salt-induced nucleic acid precipitation (Miller et al., 1988), such precipitation methods are often Known as the "salting out" method. Another example of nucleic acid isolation and/or purification includes the use of magnetic particles, to which nucleic acids can be specifically or non-specifically bound, followed by magnetic bead separation using a magnet, and washing and elution of the nucleic acids from the beads (see e.g. U.S. Patent No. 5,705,628). In some embodiments, the separation methods described above may be preceded by an enzymatic digestion step to aid in the removal of undesired proteins from the sample, such as digestion with proteinase K or other similar proteases. See, eg, US Patent No. 7,001,724. Add ribonuclease inhibitors to the lysis buffer if desired. For some cell or sample types, it may be advisable to include a protein denaturation/digestion step in the protocol. Purification methods can be used to isolate DNA, RNA, or both. When DNA and RNA are isolated together during or after the extraction process, further steps may be used to purify one or both separately. Subfractions of the extracted nucleic acid can also be generated, eg, purified by size, sequence, or other physical or chemical characteristics. In addition to the initial nucleic acid isolation step, any step in the methods of the invention may be followed by nucleic acid purification, for example to remove excess or undesired reagents, reactants or products.

Nucleic acid template molecules can be obtained as described in US Patent Application Publication No. US2002/0190663A1 published October 9, 2003. In general, nucleic acids can be extracted from biological samples by a variety of techniques, such as those described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp. 280-281 (1982). In some cases, nucleic acid can first be extracted from a biological sample and then cross-linked in vitro. In some cases, naturally associated proteins (eg, histones) can further be removed from the nucleic acid.

In other embodiments, the present invention can be readily applied to any high molecular weight double-stranded DNA, including, for example, DNA isolated from tissues, cell cultures, body fluids, animal tissues, plants, bacteria, fungi, viruses, and the like.

In some embodiments, each of the plurality of independent samples can independently comprise at least about 1 ng, 2 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 75 ng, 100 ng, 150 ng, 200 ng, 250 ng, 300 ng, 400 ng, 500 ng, 1 μg, 1.5 μg, 2 μg, 5 μg, 10 μg, 20 μg, 50 μg, 100 μg, 200 μg, 500 μg or 1000 μg or more of nucleic acid material. In some embodiments, each of the plurality of independent samples may independently comprise less than about 1 ng, 2 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 75 ng, 100 ng, 150 ng, 200 ng, 250 ng, 300 ng, 400 ng , 500 ng, 1 μg, 1.5 μg, 2 μg, 5 μg, 10 μg, 20 μg, 50 μg, 100 μg, 200 μg, 500 μg, or 1000 μg or more of nucleic acid.

In some embodiments, end repair is performed using commercial kits, such as those available from Epicentre Biotechnologies (Madison, WI), to generate blunt 5' phosphorylated nucleic acid ends.

Adapter oligonucleotides include any oligonucleotide having a sequence capable of ligation to a target polynucleotide, at least a portion of which is known. Adapter oligonucleotides may comprise DNA, RNA, nucleotide analogs, atypical nucleotides, labeled nucleotides, modified nucleotides, or combinations thereof. Adapter oligonucleotides can be single-stranded, double-stranded or partially double-stranded. Typically, a partially double-stranded linker comprises one or more single-stranded regions and one or more double-stranded regions. A double-stranded linker may comprise two different oligonucleotides (also referred to as an "oligonucleotide duplex") that hybridize to each other, and the hybridization may leave one or more blunt ends, one or more 3' overhangs, One or more 5' overhangs, one or more bulges due to mismatched and/or unpaired nucleotides, or a combination of these. In some embodiments, a single-stranded linker comprises two or more sequences capable of hybridizing to each other. When two such hybridizable sequences are contained in a single-stranded junction, hybridization results in a hairpin structure (hairpin junction). A "bubble" structure results when two hybridized regions of a linker are separated from each other by a non-hybridized region. A bubble-containing linker may consist of a single linker oligonucleotide comprising internal hybridization, or may comprise two or more linker oligonucleotides which hybridize to each other. For example, internal sequence hybridization between two hybridizable sequences within a linker can create a double-stranded structure in a single-stranded linker oligonucleotide. Different kinds of adapters can be used in combination, such as hairpin adapters and double-stranded adapters, or adapters of different sequences. The hybridizable sequence in the hairpin adapter may or may not include one or both ends of the oligonucleotide. When neither terminus is included in a hybridizable sequence, both termini are "free" or "overhanging". When only one end of the adapter is hybridizable to another sequence, the other end forms an overhang, such as a 3' overhang or a 5' overhang. When both the 5' terminal nucleotide and the 3' terminal nucleotide are contained in the hybridizable sequence such that the 5' terminal nucleotide and the 3' terminal nucleotide are complementary to each other and hybridize, the terminal is called "blunt". (blunt)". Different adapters can be ligated to the target polynucleotide in successive reactions or simultaneously. For example, a first linker and a second linker can be added to the same reaction. The linker can be treated prior to binding to the target polynucleotide. For example, terminal phosphates may be added or removed.

Adapters may contain one or more of a variety of sequence elements, including, but not limited to, one or more amplification primer annealing sequences or their complements, one or more sequencing primer annealing sequences or their complements, one or more A barcode sequence, one or more consensus sequences shared among a plurality of different adapters or a subset of different adapters, one or more restriction enzyme recognition sites, one or more overhangs complementary to one or more target polynucleotides Multiple overhangs, one or more probe binding sites (e.g. for attachment to sequencing platforms, e.g. flow cells for massively parallel sequencing, e.g. developed by Illumina), one or more random or nearly random sequence (e.g., one or more nucleotides randomly selected at one or more positions from a set of two or more different nucleotides, wherein one or more of the different nucleotides selected at one or more positions each present in the pool of adapters containing the random sequence), and combinations thereof. Two or more sequence elements may be non-adjacent to each other (eg, separated by one or more nucleotides), adjacent to each other, partially overlapping, or completely overlapping. For example, an amplification primer annealing sequence can also be used as a sequencing primer annealing sequence. The sequence element can be at or near the 3' end, at or near the 5' end, or within the adapter oligonucleotide. When the linker oligonucleotide is capable of forming a secondary structure (such as a hairpin), the sequence element may be partially or completely external to the secondary structure, partially or completely internal to the secondary structure, or participate in the secondary structure. Between sequences of structures. For example, when an adapter oligonucleotide comprises a hairpin structure, sequence elements may be located partially or completely inside or outside the hybridizable sequence ("stem"), including sequences between the hybridizable sequences (" loop (loop)"). In some embodiments, the first linker oligonucleotides of the plurality of first linker oligonucleotides having different barcode sequences comprise sequence elements that are common to the ensemble of the plurality of first linker oligonucleotides. In some embodiments, all second adapter oligonucleotides comprise a sequence element common among all second adapter oligonucleotides that is different from the consensus sequence element shared by the first adapter oligonucleotide. The difference in sequence elements may be any difference that results in incomplete alignment of at least some of the different linkers, for example due to a change in sequence length, deletion or insertion of one or more nucleotides, or nucleotide composition within one or more nucleosides Changes in acid sites (eg, base changes or base modifications). In some embodiments, an adapter oligonucleotide comprises a 5' overhang, a 3' overhang, or both, that are complementary to one or more target polynucleotides. Complementary overhangs can be one or more nucleotides in length, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides. For example, complementary overhangs can be about 1, 2, 3, 4, 5 or 6 nucleotides in length. Complementary overhangs can include fixed sequences. A complementary overhang may comprise a random sequence of one or more nucleotides such that one or more nucleotides are randomly selected at one or more positions from a set of two or more different nucleotides, in Each of the different nucleotides selected by one or more sites is present in a pool of adapters having complementary overhangs comprising the random sequence. In some embodiments, the adapter overhang is complementary to a target polynucleotide overhang produced by restriction endonuclease digestion. In some embodiments, the linker overhang comprises adenine or thymine.

Adapter oligonucleotides may be of a suitable length at least sufficient to accommodate the sequence element or elements they contain. In some embodiments, the linker is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 80, 90, 100, 200 or more nucleotides, or less than that length, or more than that length. In some embodiments, linkers may be about 10 to about 50 nucleotides in length. In further embodiments, the linker may be about 20 to about 40 nucleotides in length.

As used herein, the term "barcode" refers to a known nucleic acid sequence that allows identification of certain features of the polynucleotide associated with the barcode. In some embodiments, the polynucleotide to be identified is characterized by the sample from which the polynucleotide was derived. In some embodiments, the barcode length can be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides. For example, the barcode can be at least 10, 11, 12, 13, 14 or 15 nucleotides in length. In some embodiments, barcodes may be less than 10, 9, 8, 7, 6, 5, or 4 nucleotides in length. For example, barcodes can be less than 10 nucleotides in length. In some embodiments, barcodes associated with certain polynucleotides are of different lengths than barcodes associated with other polynucleotides. Typically, the barcodes are of sufficient length and contain sufficiently distinct sequences to allow identification of samples based on their associated barcodes. In some embodiments, one or more nucleotide mutations, insertions or deletions in the barcode sequence, for example, 1, 2, 3, 4, 5, 6, 7, 8 After mutations, insertions, or deletions of 1, 9, 10, or more nucleotides, the barcodes and their associated sample origins are accurately identified. In some embodiments, 1, 2 or 3 nucleotides may be mutated, inserted and/or deleted. In some embodiments, each barcode in the plurality of barcodes is at least two nucleotide positions, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more positions that differ from every other barcode in multiple barcodes. In some embodiments, each barcode can differ from every other barcode in at least 2, 3, 4, or 5 positions. In some embodiments, both the first locus and the second locus comprise at least one of the plurality of barcode sequences. In some embodiments, the barcode for the second site is selected independently of the barcode for the first adapter oligonucleotide. In some embodiments, the first locus and the second locus having a barcode are paired such that the paired sequences comprise the same or different one or more barcodes. In some embodiments, the methods of the invention further comprise identifying the sample from which the target polynucleotide was derived based on the barcode sequence linked to the target polynucleotide. Typically, a barcode may comprise a nucleic acid sequence that, when linked to a target polynucleotide, serves as an identifier for the sample from which the target polynucleotide was derived.

In eukaryotes, genomic DNA is packaged into chromatin to organize chromosomes within the nucleus. The basic structural unit of chromatin is the nucleosome, which consists of 146 base pairs (bp) of DNA wrapped around a histone octamer. The histone octamer consists of two copies of each of the core histone H2A-H2B dimers and H3-H4 dimers. Nucleosomes are regularly spaced along the DNA in what is commonly referred to as "beads of rope".

Chaperone proteins and associated assembly factors mediate the assembly of core histones and DNA into nucleosomes. Almost all of these factors are core histone binding proteins. Some histone chaperones, such as nucleosome assembly protein-1 (NAP-1), show a preference for binding to histones H3 and H4. It has also been observed that newly synthesized histones are acetylated and then deacetylated after assembly into chromatin. Factors that mediate histone acetylation or deacetylation thus play an important role in the process of chromatin assembly.

Generally, two in vitro methods have been developed for remodeling or assembling chromatin. One method is ATP-independent, while the second is ATP-dependent. An ATP-independent approach for remodeling chromatin involves DNA and core histones, coupled with NAP-1-like proteins or salts, that act as histone chaperones. This approach results in a random arrangement of histones on the DNA, which in essence does not accurately mimic the natural core nucleosomal particles in the cell. These particles are often referred to as mononucleosomes because they are irregularly arranged, using extended nucleosome arrays and DNA sequences that are often no longer than 250 bp (Kundu, T.K. et al., Mol. Cell 6:551-561, 2000) . To generate extended arrays of ordered nucleosomes on longer DNA sequences, chromatin must be assembled by an ATP-dependent process.

ATP-dependent assembly of periodic nucleosome arrays resembles that seen in native chromatin, requiring DNA sequences, core histone granules, chaperone proteins, and ATP-utilizing chromatin assembly factors. ACF (ATP-utilizing Chromatin Assembly and Remodeling Factor) or RSF (Remodeling and Spacing Factor) are two widely studied assembly factors used to generate extended, ordered arrays of nucleosomes into chromatin in vitro (Fyodorov, D.V. and Kadonaga, J.T. Method Enzymol. 371:499-515, 2003; Kundu, T.K. et al., Mol. Cell 6:551-561, 2000).

In particular embodiments, the methods of the present invention can be readily applied to any type of fragmented double-stranded DNA, including but not limited to, for example, cell-free DNA isolated from plasma, serum and/or urine; isolated from cells and /or apoptotic DNA of tissues; DNA fragmented enzymatically in vitro (e.g., by deoxyribonuclease I and/or restriction endonucleases); and/or DNA fragmented mechanically (fluid-filled shears) cutting, sonication, nebulization, etc.).

Nucleic acids obtained from biological samples can be fragmented to produce suitable fragments for analysis. Template nucleic acids can be fragmented or sheared to a desired length using a variety of mechanical, chemical and/or enzymatic methods. DNA can be randomly sheared by sonication, eg, the Covaris method, short-term exposure to deoxyribonucleases, or using a mixture of one or more restriction enzymes or a transposase or nickase. RNA can be fragmented by short-term exposure to ribonucleases, heat plus magnesium, or by shearing. RNA can be converted to cDNA. If fragmentation is used, the RNA can be converted to cDNA either before or after fragmentation. In some embodiments, nucleic acids from a biological sample are fragmented by sonication. In other embodiments, nucleic acids are fragmented by a fluid-filled shearing instrument. Typically, each nucleic acid template molecule can range from about 2 kb bases to about 40 kb. In various embodiments, nucleic acids may be fragments of about 6kb-10kb. Nucleic acid molecules can be single-stranded, double-stranded, or double-stranded with single-stranded regions (eg, a stem-loop structure).

In some embodiments, the cross-linked DNA molecules can be subjected to a size selection step. Size selection of nucleic acids can be performed on cross-linked DNA molecules below or above a specific size. Size selection can further be influenced by cross-linking frequency and/or fragmentation method, for example by choosing frequent-cutting restriction enzymes or rare-cutting restriction enzymes. In some embodiments, compositions can be prepared comprising DNA molecules crosslinked in the range of about 1 kb to 5 Mb, about 5 kb to 5 Mb, about 5 kb to 2 Mb, about 10 kb to 2 Mb, about 10 kb to 1 Mb, about 20 kb to 1 Mb , about 20kb to 500kb, about 50kb to 500kb, about 50kb to 200kb, about 60kb to 200kb, about 60kb to 150kb, about 80kb to 150kb, about 80kb to 120kb, or about 100kb to 120kb or defined by any of these values Any range of (eg, about 150 kb to 1 Mb).

In some embodiments, sample polynucleotides are fragmented into one or more populations of fragmented DNA molecules of a particular size range. In some embodiments, from at least about 1, about 2, about 5, about 10, about 20, about 50, about 100, about 200, about 500, about 1000, about 2000, about 5000, about 10,000, about 20,000 , about 50,000, about 100,000, about 200,000, about 500,000, about 1,000,000, about 2,000,000, about 5,000,000, about 10,000,000 or more genome equivalents of starting DNA to generate fragments. Fragmentation can be accomplished by methods known in the art, including chemical, enzymatic and mechanical fragmentation. In some embodiments, the fragments have an average length of about 10 to about 10,000, about 10 to about 20,000, about 10 to about 30,000, about 10 to about 40,000, about 10 to about 50,000 , about 10 to about 60,000, about 10 to about 70,000, about 10 to about 80,000, about 10 to about 90,000, about 10 to about 100,000, about 10 to about 150,000, about 10 to about 200,000, about 10 to about 300,000, about 10 to about 400,000, about 10 to about 500,000, about 10 to about 600,000, about 10 to about 700,000, about 10 to about 800,000, about 10 to about 900,000, about 10 to about 1,000,000, about 10 to about 2,000,000, about 10 to about 5,000,000, about 10 to about 10,000,000 or more nucleosides acid. In some embodiments, the average length of the fragments is from about 1 kb to about 10 Mb. In some embodiments, the average length of the fragments is from about 1 kb to 5 Mb, about 5 kb to 5 Mb, about 5 kb to 2 Mb, about 10 kb to 2 Mb, about 10 kb to 1 Mb, about 20 kb to 1 Mb, about 20 kb to 500 kb, about 50 kb to 500kb, about 50kb to 200kb, about 60kb to 200kb, about 60kb to 150kb, about 80kb to 150kb, about 80kb to 120kb, or about 100kb to 120kb or any range defined by any of these values (e.g. about 60 to 120kb) . In some embodiments, the average length of the fragments is less than about 10 Mb, less than about 5 Mb, less than about 1 Mb, less than about 500 kb, less than about 200 kb, less than about 100 kb, or less than about 50 kb. In other embodiments, the average length of the fragments is greater than about 5 kb, greater than about 10 kb, greater than about 50 kb, greater than about 100 kb, greater than about 200 kb, greater than about 500 kb, greater than about 1 Mb, greater than about 5 Mb, or greater than about 10 Mb. In some embodiments, mechanically accomplished fragmentation comprises sonicating the sample DNA molecules. In some embodiments, fragmenting comprises treating the sample DNA molecule with one or more enzymes under conditions suitable for the one or more enzymes to generate double-stranded nucleic acid breaks. Examples of enzymes used to generate DNA fragments include sequence-specific and non-sequence-specific nucleases. Non-limiting examples of nucleases include DNase I, Fragmentase, restriction endonuclease, variants thereof, and combinations thereof. For example, digestion with DNase I can involve the absence of Mg ⁺⁺ and the presence of Mn ⁺⁺ , causing random double-strand breaks in the DNA. In some embodiments, fragmenting comprises treating the sample DNA molecules with one or more restriction endonucleases. Fragmentation can generate fragments with 5' overhangs, 3' overhangs, blunt ends, or combinations thereof. In some embodiments, eg, when fragmentation comprises the use of one or more restriction endonucleases, cleavage of the sample DNA molecule leaves a predictable sequence. In some embodiments, the method includes the step of selecting the size of the fragments by standard methods, such as column purification or separation from an agarose gel.

In some embodiments, the 5' and/or 3' terminal nucleotide sequences of the fragmented DNA are not modified prior to the ligation reaction. For example, fragmentation by restriction endonucleases can be used to leave predictable overhangs, which are then ligated with nucleic acid ends containing overhangs that are complementary to predictable overhangs on the DNA fragments. In another example, enzymatic cleavage leaves predictable blunt ends, and the blunt-ended DNA fragments can subsequently be ligated to nucleic acids comprising blunt ends, such as adapters, oligonucleotides, or polynucleotides. In some embodiments, the fragmented DNA molecules are blunt-end modified (or "end repaired") so that DNA fragments are produced with blunt ends prior to ligation to adapters. The blunt-end modification step can be accomplished by incubation with a suitable enzyme, such as a DNA polymerase having both 3' to 5' exonuclease activity and 5' to 3' exonuclease activity, such as T4 polymerase. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 may be added after end repair or more nucleotides, such as one or more adenines, one or more thymines, one or more guanines, or one or more cytosines, to create an overhang. For example, 1, 2, 3, 4, 5 or 6 nucleotides may be added after pairing of the ends. A DNA fragment having an overhang can be ligated to one or more nucleotides having a complementary overhang, such as an oligonucleotide, an adapter oligonucleotide or a polynucleotide, eg in a ligation reaction. For example, a template-independent polymerase can be used to add a single adenine to the 3' end of the end-repaired DNA fragment, which is then ligated to one or more adapters, each adapter having a thymine at the 3' end. In some embodiments, a nucleic acid, such as an oligonucleotide or polynucleotide, can be added to a blunt-ended double-stranded DNA molecule that has been modified by one or more nucleotide extensions at the 3' end, and subsequently subjected to 5' ' Phosphorylation. In some cases, extension of the 3' end can be accomplished with a polymerase such as Klenow polymerase or any suitable polymerase provided herein, or in the presence of one or more dNTPs in a suitable buffer containing magnesium In the case of terminal deoxynucleotidyl transferase. In some embodiments, target polynucleotides with blunt ends are ligated to one or more adapters containing blunt ends. Phosphorylation of the 5' end of the DNA fragment molecule can be performed, for example, with T4 polynucleotide kinase in a suitable buffer containing ATP and magnesium. Fragmented DNA molecules may optionally be treated to dephosphorylate the 5' or 3' ends, for example by using enzymes known in the art, such as phosphatases.

The term "ligation" as used herein in relation to two polynucleotides (e.g., an adapter oligonucleotide and a target polynucleotide), means the covalent joining of two separate DNA fragments to produce a single DNA with a continuous backbone. Larger polynucleotides. Methods for joining two DNA fragments are known in the art and include, but are not limited to, enzymatic and non-enzymatic (eg, chemical) methods. Examples of non-enzymatic ligation reactions include the non-enzymatic ligation techniques described in US Patent Nos. 5,780,613 and 5,476,930, the contents of which are incorporated herein by reference. In some embodiments, the adapter oligonucleotide is ligated to the target polynucleotide by a ligase, such as DNA ligase or RNA ligase. A variety of ligases are known in the art, each with characterized reaction conditions, and include, but are not limited to, NAD ⁺ -dependent ligases, including tRNA ligase, Taq DNA ligase, Thermus filiformis DNA Ligase, E. coli DNA Ligase, Tth DNA Ligase, Thermus scotoductus DNA Ligase (Type I and Type II), Thermostable Ligase, Ampligase Thermostable DNA Ligase, VanC Type Ligase , 9°N DNA ligase, Tsp DNA ligase and novel ligases discovered through bioprospecting; ATP-dependent ligases include T4RNA ligase, T4DNA ligase, T3DNA ligase, T7DNA ligase, Pfu DNA ligase, DNA Ligase I, DNA ligase III, DNA ligase IV, and novel ligases discovered through bioprospecting; and their wild-type, mutant isoforms, and genetically engineered variants.

Ligation can occur between DNA fragments having hybridizable sequences, such as complementary overhangs. Ligation can also occur between two blunt ends. Typically, a 5' phosphate group is utilized in the ligation reaction. The 5' phosphate is provided by the target polynucleotide, the linker oligonucleotide, or both. A 5' phosphate group can be added to or removed from the DNA fragments to be ligated as necessary. Methods of adding or removing 5' phosphate groups to or from 5' phosphate groups are known in the art and include, but are not limited to, enzymatic and chemical methods. Enzymes for adding and/or removing 5' phosphate groups include kinases, phosphatases and polymerases. In some embodiments, both ends ligated in a ligation reaction (e.g., an adapter end and a target polynucleotide end) provide a 5' phosphate group such that two covalent linkages are created during ligation of the two ends. In some embodiments, only one of the two ends joined in the ligation reaction (e.g., only one of the adapter end and the target polynucleotide end) contributes the 5' phosphate so that only one of the two ends is produced during ligation. a covalent link.

In some embodiments, only one strand is ligated to the adapter oligonucleotide at one or both ends of the target polynucleotide. In some embodiments, both strands are ligated to an adapter oligonucleotide at one or both ends of the target polynucleotide. In some embodiments, the 3' phosphate is removed prior to ligation. In some embodiments, an adapter oligonucleotide is added to both ends of a target polynucleotide, with one or both strands ligated to the adapter oligonucleotide at each end. When both strands are ligated to adapter oligonucleotides at both ends, ligation can be followed by a cleavage reaction that leaves a 5' overhang that can serve as a template for the extension of the corresponding 3' end, which may or may not include One or more nucleotides derived from an adapter oligonucleotide. In some embodiments, one end of the target polynucleotide is ligated to a first adapter oligonucleotide, and the other end is ligated to a second adapter oligonucleotide. In some embodiments, both ends of a target polynucleotide are ligated to opposite ends of a single adapter oligonucleotide. In some embodiments, the linker oligonucleotide to which the target polynucleotide is ligated comprises blunt ends. In some embodiments, each sample can be subjected to a separate ligation reaction, using a different first adapter oligonucleotide, the first adapter oligonucleotide comprising at least one barcode sequence for each sample such that neither The barcode sequences are linked to target polynucleotides of more than one sample. A DNA fragment or target polynucleotide having an adapter oligonucleotide attached is said to be "tagged" with the adapter ligated.

In some cases, the ligation reaction can be performed at about 0.1 ng/μL, about 0.2 ng/μL, about 0.3 ng/μL, about 0.4 ng/μL, about 0.5 ng/μL, about 0.6 ng/μL, about 0.7 ng/μL , about 0.8ng/μL, about 0.9ng/μL, about 1.0ng/μL, about 1.2ng/μL, about 1.4ng/μL, about 1.6ng/μL, about 1.8ng/μL, about 2.0ng/μL, about 2.5ng/μL, about 3.0ng/μL, about 3.5ng/μL, about 4.0ng/μL, about 4.5ng/μL, about 5.0ng/μL, about 6.0ng/μL, about 7.0ng/μL, about 8.0ng /μL, about 9.0ng/μL, about 10ng/μL, about 15ng/μL, about 20ng/μL, about 30ng/μL, about 40ng/μL, about 50ng/μL, about 60ng/μL, about 70ng/μL, about 80ng/μL, about 90ng/μL, about 100ng/μL, about 150ng/μL, about 200ng/μL, about 300ng/μL, about 400ng/μL, about 500ng/μL, about 600ng/μL, about 800ng/μL or about 1000ng/μL DNA fragment or target polynucleotide concentration. For example, ligation can be performed at a DNA fragment or target polynucleotide concentration of about 100 ng/μL, about 150 ng/μL, about 200 ng/μL, about 300 ng/μL, about 400 ng/μL, or about 500 ng/μL.

In some cases, the ligation reaction can be performed at about 0.1 to 1000 ng/μL, about 1 to 1000 ng/μL, about 1 to 800 ng/μL, about 10 to 800 ng/μL, about 10 to 600 ng/μL, about 100 to 600 ng/μL or about 100 to 500 ng/μL of DNA fragment or target polynucleotide concentration.

In some cases, the ligation reaction can be performed for greater than about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3 hours, About 4 hours, about 5 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, or about 96 hours. In other cases, the ligation reaction can be performed in less than about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3 hours , about 4 hours, about 5 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, or about 96 hours. For example, the ligation reaction can be performed for about 30 minutes to 90 minutes. In some embodiments, an adapter is ligated to a target polynucleotide resulting in a ligation product polynucleotide having a 3' overhang comprising a nucleotide sequence derived from the adapter.

In some embodiments, after ligation of at least one adapter oligonucleotide to the target polynucleotide, the 3' end of the one or more target polynucleotides is extended using the one or more ligated adapter oligonucleotides as a template . For example, an adapter comprising two hybridizing oligonucleotides is ligated only to the 5' end of the target polynucleotide, which allows the unligated 3' end of the target to be extended using the ligated strand of the adapter as a template, simultaneously or subsequently displacing the unligated chain. The two strands of the adapter containing the two hybridizing oligonucleotides can be ligated to the target polynucleotide so that the ligated product has a 5' overhang, and the complementary 3' end can be extended using the 5' overhang as a template. As a further example, a hairpin adapter oligonucleotide can be ligated to the 5' end of the target polynucleotide. In some embodiments, the 3' end of the extended target polynucleotide comprises one or more nucleotides from an adapter oligonucleotide. For target polynucleotides with adapters attached to both ends, both 3' ends of a double-stranded target polynucleotide with a 5' overhang can be extended. This 3' end extension, or "fill-in reaction", generates a complementary sequence or adapter oligonucleotide template "complement" that hybridizes to the template, thereby filling in the 5' overhang, creating a double chain sequence region. When both ends of the double stranded target polynucleotide have 5' overhangs, the 5' overhangs are filled in by extension of the 3' ends of the complementary strands and the product is fully double stranded. Extension can be performed by any suitable polymerase known in the art, such as DNA polymerase, a variety of which are commercially available. A DNA polymerase may comprise DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity, or DNA-dependent and RNA-dependent DNA polymerase activity. DNA polymerases can be thermostable or non-thermostable. Examples of DNA polymerases include, but are not limited to, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Sso Polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerase, Platinum Taq polymerase Enzyme, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Klenow fragment, and its variants, modified products and derivatives. 3' end extension can be performed before or after pooling of target polynucleotides from independent samples.

In certain embodiments, the present invention provides methods for enriching and analyzing target nucleic acids. In some cases, the method used for enrichment is a solution-based format. In some cases, a target nucleic acid can be labeled with a label. In other cases, a target nucleic acid can be cross-linked with one or more associated molecules labeled with a label. Examples of labels include, but are not limited to, biotin, polyhistidine tags, and chemical tags (such as alkynes and azides used in Click Chemistry methods). In addition, labeled target nucleic acids can be captured using a capture agent and thus enriched. Capture agents can be streptavidin and/or avidin, antibodies, chemical moieties (e.g., alkynes, azides), and any biological, chemical, or other known in the art for affinity purification. , physical or enzymatic reagents.

In some cases, immobilized or non-immobilized nucleic acid probes can be used to capture target nucleic acids. Target nucleic acids are enriched from a sample, eg, by hybridization with probes on a solid support or in solution. In some embodiments, a sample may be a genomic sample. In some embodiments, the probes may be amplicons. An amplicon may comprise a predetermined sequence. In addition, hybridized target nucleic acids can be washed away and/or the probes can be eluted. A target nucleic acid can be a DNA, RNA, cDNA or mRNA molecule.

In some cases, the enrichment method can include contacting a sample containing the target nucleic acid with a probe, binding the target nucleic acid to a solid support. In some cases, a sample can be fragmented using chemical, physical, or enzymatic methods to produce target nucleic acids. In some cases, a probe can specifically hybridize to a target nucleic acid. In some cases, the average size of the target nucleic acid can be about 50 to 5000, about 50 to 2000, about 100 to 2000, about 100 to 1000, about 200 to 1000, about 200 to 800, or about 300 to 800, about 300 to 600, or about 400 to 600 nucleotide residues. Target nucleotides can be further separated from non-bound nucleic acids in the sample. The solid support can be washed and/or eluted to obtain enriched target nucleic acids. In some embodiments, the enrichment step may be repeated about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. For example, the enrichment step can be repeated about 1, 2 or 3 times.

In some cases, the enrichment method can include providing amplicons derived from probes attached to a solid support for amplification. A solid support may include nucleic acid probes immobilized on the support to capture specific target nucleic acids from a sample. Amplicons derived from the probes can hybridize to the target nucleic acid. After hybridization to the probe amplicon, the target nucleic acid in the sample can be enriched by capturing (e.g., by a capture agent such as biotin, antibody, etc.) and washing and/or eluting the hybridized target nucleic acid from the captured probe ( Figure 4). The target nucleic acid sequence is further amplified using, for example, PCR methods to generate an amplified pool enriched in PCR products.

In some cases, the solid support can be a microarray, slide, chip, microwell, column, tube, particle or bead. In some embodiments, the solid support can be coated with streptavidin and/or avidin. In other embodiments, the solid support can be coated with antibodies. In addition, solid supports may comprise glass, metal, ceramic or polymeric materials. In some embodiments, the solid support can be a nucleic acid microarray (eg, a DNA microarray). In other embodiments, the solid support can be paramagnetic beads.

In some cases, the enrichment method can include digestion with a second restriction endonuclease, self-ligation (eg, self-circularization), and re-digestion with the original restriction endonuclease. In specific embodiments, only ligation products will be linearized and available for adapter ligation and sequencing. In other cases, ligation of the junction sequence itself can be used for hybridization-based enrichment using bait-probes complementary to the junction sequence.

In certain embodiments, the present invention provides methods for amplifying enriched DNA. In some cases, the enriched DNA is a read pair. Read pairs can be obtained by the method of the present invention.

In some embodiments, one or more amplification and/or replication steps are used to prepare a library to be sequenced. Any amplification method known in the art can be used. Examples of amplification techniques that may be used include, but are not limited to, quantitative polymerase chain reaction, quantitative fluorescent polymerase chain reaction (QF-PCR), multiplex fluorescent polymerase chain reaction (MF-PCR), real-time polymerase chain reaction reaction (RTPCR), single cell PCR, restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP), PCK-RFLPIRT-PCR-IRFLP, hot start PCR, nested polymerization Enzyme chain reaction, in situ PCR, in situ rolling circle amplification (RCA), bridge PCR, ligation-mediated PCR, Qb replicase amplification, reverse polymerase Chain reaction, picotiter polymerase chain reaction and emulsion polymerase chain reaction. Other suitable amplification methods include ligase chain reaction (LCR), transcriptional amplification, autonomous sequence replication, selective amplification of target polynucleotide sequences, consensus primer polymerase chain reaction (CP-PCR), random Primed-polymerase chain reaction (AP-PCR), degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), and nucleic acid-based sequence amplification (NABSA). Other amplification methods useful herein include those described in US Patent Nos. 5,242,794, 5,494,810, 4,988,617, and 6,582,938.

In a particular embodiment, after the DNA molecules are partitioned into partitions, PCR is used to amplify the DNA molecules. In some cases, one or more specific promoter sequences within the amplification adapter are used for PCR amplification. Amplification adapters can be ligated to the fragmented DNA molecules either before or after partitioning into individual partitions. Polynucleotides comprising amplification adapters having suitable promoter sequences at both ends can be exponentially amplified by PCR. For example, polynucleotides with only one suitable promoter sequence can only be linearly amplified due to defective ligation efficiency of the amplification adapter containing the promoter sequence. Further, polynucleotides may be excluded altogether from amplification (eg, PCR amplification) if adapters containing appropriate promoter sequences are not ligated. In some embodiments, the number of PCR cycles varies between 10-30, but can be as low as 9, 8, 7, 6, 5, 4, 3, 2 or less or as high as 40, 45, 50, 55 , 60 or more. Thus, following PCR amplification, fragments loaded with an amplified adapter with a suitable promoter sequence that can be amplified exponentially can be amplified at a much higher (1000 times or more) concentration exists. Benefits of PCR include, but are not limited to, more consistent sequence coverage compared to whole-genome amplification techniques such as amplification with random primers or multiple displacement amplification using phi29 polymerase—since each fragment Can only be copied at most once and due to controlled amplification by thermal cycling procedures; the formation rate of chimeric molecules is substantially lower than that of, for example, MDA (Lasken et al., 2007, BMC Biotechnology) - due to chimeric molecules displaying non- Biological sequences, which pose significant challenges to accurate sequence assembly, which can lead to higher rates of misassembly or highly ambiguous and fragmented assemblies; reduced sequence-specific biases that can be due to the incorporation of randomized primers commonly used in MDA , which is in contrast to the use of specific primer sites with specific sequences; the reproducibility of the amount of final amplified DNA product is more reproducible, which is controlled by the selection of the number of PCR cycles; Compared with genome amplification techniques, replication with polymerases commonly used in PCR provides higher fidelity.

In some embodiments, one or more target polynucleotides are amplified after, or as part of, a fill-in reaction using a first primer and a second primer, wherein the first primer comprises a A sequence to which at least a portion of the complement of the one or more first adapter oligonucleotides hybridizes, and further wherein the second primer comprises a sequence to which at least a portion of the complement of the one or more second adapter oligonucleotides hybridizes. Each of the first primer and the second primer can have a suitable length, for example about, about less than or about more than 10, 15, 20, 25, 30, 35, 40, 45 , 50, 55, 60, 65, 70, 75, 80, 90, 100 or more nucleotides, any part or all of which may be complementary to the corresponding target sequence (e.g., about , less than about or greater than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides). For example about 10 to 50 nucleotides may be complementary to the corresponding target sequence.

"Amplification"refers to any method of increasing the number of copies of a target sequence. In some cases, a replication reaction may produce only a single complementary copy/replica of the polynucleotide. Methods for primer-directed amplification of target polynucleotides are known in the art, including but not limited to polymerase chain reaction (PCR)-based methods. Favorable conditions for PCR amplification of target sequences are known in the art, can be optimized at various steps in the method, and depend on the characteristics of the elements in the reaction, such as target type, target concentration, length of sequence to be amplified, target and/or or the sequence of one or more primers, primer length, primer concentration, polymerase used, reaction volume, ratio of one or more elements to one or more other elements, and others, some or all of which may be determined by Change. Generally, PCR involves the steps of denaturation of the target to be amplified (if double-stranded), hybridization of one or more primers to the target, and primer extension by a DNA polymerase, which are repeated (or "cycled") to amplify Amplified target sequence. Steps in the method can be optimized for various outcomes, eg, to increase yield, reduce formation of spurious products, and/or increase or decrease specificity of primer annealing. Optimization methods are known in the art and include adjusting the type or number of elements in the amplification reaction and/or the conditions of a given step in the method, such as the temperature of a particular step, the duration of a particular step and/or the number of cycles.

In some embodiments, the amplification reaction may comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 , 100, 150, 200 or more cycles. In some embodiments, an amplification reaction may comprise at least about 20, 25, 30, 35, or 40 cycles. In some embodiments, the amplification reaction may comprise up to about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more cycles. A cycle may contain any number of steps, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more steps. A step may comprise any temperature or temperature gradient suitable to achieve the purpose of a given step, including but not limited to 3' end extension (e.g., adapter fill-in), primer annealing, primer extension, and strand denaturation. A step can last for any amount of time, including but not limited to about, about less than, or about more than 1 second, 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds , 55 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 120 seconds, 180 seconds, 240 seconds, 300 seconds, 360 seconds, 420 seconds, 480 seconds, 540 seconds, 600 seconds, 1200 seconds, 1800 seconds seconds or more, including indefinitely, until manually interrupted. Any number of cycles containing different steps can be combined in any order. In some embodiments, the combination comprises different cycles of different steps such that the total number of cycles in the combination is about, about less than, or about greater than 5, 10, 15, 20, 25, 30, 35 , 40, 50, 60, 70, 80, 90, 100, 150, 200 or more cycles. In some embodiments, the amplification is performed after the fill-in reaction.

In some embodiments, the amplification reaction can be at least about 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, 12 ng, 14 ng, 16 ng, 18 ng, 20 ng, 25 ng, 30 ng, 40 ng, 50 ng , 100ng, 200ng, 300ng, 400ng, 500ng, 600ng, 800ng, 1000ng of target DNA molecules. In other embodiments, the amplification reaction can be performed at less than about 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, 12 ng, 14 ng, 16 ng, 18 ng, 20 ng, 25 ng, 30 ng, 40 ng, 50ng, 100ng, 200ng, 300ng, 400ng, 500ng, 600ng, 800ng, 1000ng of target DNA molecules.

Amplification can be performed before or after pooling of target polynucleotides from independent samples.

The methods of the invention involve determining the amount of amplifiable nucleic acid present in a sample. Any known method can be used to quantify the amplifiable nucleic acid, an exemplary method is polymerase chain reaction (PCR), specific quantitative polymerase chain reaction (qPCR). qPCR is a polymerase chain reaction-based technique for amplifying and simultaneously quantifying target nucleic acid molecules. qPCR allows detection and quantification (either as absolute number of copies or relative quantity when normalized to DNA input or another normalizing gene) of specific sequences in a DNA sample. The protocol follows the general principles of the polymerase chain reaction, with the additional feature of real-time quantification of amplified DNA as it accumulates in the reaction after each amplification cycle. QPCR is, for example, described in Kurnit et al. (US Patent No. 6,033,854), Wang et al. (US Patent Nos. 5,567,583 and 5,348,853), Ma et al. (The Journal of American Science, 2(3), 2006), Heid et al. (Genome Research 986-994, 1996) , Sambrook and Russell (Quantitative PCR, Cold Spring Harbor Protocols, 2006), and Higuchi (US Patent Nos. 6,171,785 and 5,994,056). These contents are incorporated herein by reference in their entirety.

Other quantitative methods include the use of fluorescent dyes that intercalate into double-stranded DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized to complementary DNA. These methods are broadly applicable and also specifically applicable to real-time PCR, which are further detailed as examples. In the first method, a DNA-binding dye binds to all double-stranded (ds) DNA in PCR, causing a fluorescent reaction of the dye. Thus, an increase in DNA product during PCR increases the fluorescence intensity and is measured at each cycle, allowing the DNA concentration to be quantified. The reaction was performed similarly to standard PCR, with the addition of fluorescent (ds) DNA dye. The reaction is run in a thermal cycler, and after each cycle, the level of fluorescence is measured with a detector; the dye only fluoresces when bound to (ds)DNA (ie the PCR product). The concentration of (ds)DNA in PCR can be determined according to standard dilution methods. As with other real-time PCR methods, the values obtained have no absolute units associated with them. Comparing measured DNA/RNA samples and standard dilutions yields a sample-to-standard fraction or ratio, allowing relative comparisons between different tissues or experimental conditions. To ensure accurate quantification and/or expression of target genes, normalization to stably expressed genes can be performed. The copy number of the unknown gene can likewise be normalized to the gene of known copy number.

The second method uses sequence-specific RNA- or DNA-based probes to quantify DNA containing only the probe sequence; thus, the use of reporter probes greatly improves specificity and allows for the quantification of DNA even in the presence of some non-specific DNA amplification. Quantitative time increase. This allows multiplexing, that is, the analysis of several genes in the same reaction by using specific probes with different color labels, provided that all genes are amplified with similar efficiency.

This method is usually performed with a DNA-based probe with a fluorescent reporter molecule (such as 6-carboxyfluorescein) on one end and a fluorescence quencher molecule (such as 6-carboxy-tetramethylrhodamine) on the other end of the probe. ). The reporter molecule is in close proximity to the quencher molecule, which prevents its fluorescence from being detected. The 5' to 3' exonuclease activity of a polymerase (eg, Taq polymerase) cleaves the probe, breaking the reporter-quencher proximity, allowing unquenched fluorescence to be detected. The increase in the product of reporter probe targeting at each PCR cycle results in a proportional increase in fluorescence due to breakdown of the probe and release of the reporter molecule. The reaction is performed similarly to a standard PCR reaction, adding the reporter probe. As the reaction begins, both probes and primers anneal to the DNA target during the annealing phase of PCR. Polymerization of a new DNA strand begins with the primer, and once the polymerase reaches the probe, its 5'-3' exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence. Fluorescence was detected and measured in a real-time PCR thermal cycler, and the geometric growth of fluorescence corresponding to the exponential growth of the product was used to determine the threshold cycle in each reaction.

The relative concentration of DNA present during the exponential phase of the reaction was determined by plotting fluorescence versus cycle number on a logarithmic scale (so exponential growth would yield a straight line). Determine the threshold used to detect fluorescence over the background. The number of cycles at which the fluorescence from the sample reaches the threshold is referred to as the threshold cycle _Ct . Since the amount of DNA doubles every cycle during the exponential phase, the relative amount of DNA can be calculated, eg, a sample with a _Ct 3 cycles earlier than another sample has 2 ³ =8 times more template. The amount of nucleic acid (e.g. RNA or DNA) is then determined by comparing the results to a standard curve by serial dilution (e.g. undiluted, 1:4, 1:16, 1:64) of known amounts of nucleic acid real-time PCR generation.

In certain embodiments, the qPCR reaction involves a dual fluorophore approach utilizing fluorescence resonance energy transfer (FRET), such as LIGHTCYCLER hybridization probes, in which two oligonucleotide probes anneal to the amplicon (see e.g. US Patent No. 6,174,670). Oligonucleotides are designed to hybridize in a head-to-tail orientation, with fluorophores separated by a distance compatible with efficient energy transfer. Examples of other labeled oligonucleotides constructed to signal when bound to nucleic acid or incorporated into extension products include: SCORPIONS probes (e.g., Whitcombe et al., Nature Biotechnology 17:804-807, 1999, and U.S. Patent No. 6,326,145), Sunrise (or AMPLIFLOUR) primers (such as Nazarenko et al., Nuc.Acids Res.25:2516-2521, 1997, and U.S. Patent No. 6,117,635), and LUX primers and MOLECULAR BEACONS probes (such as Tyagi et al., Nature Biotechnology 14:303-308 , 1996 and US Patent No. 5,989,823).

In other embodiments, the qPCR reaction uses a fluorescent Taqman method and an instrument capable of measuring fluorescence in real time (eg, an ABI Prism 7700 Sequence Detector). The hybridization probes used in the Taqman reaction are labeled with two different fluorescent dyes. One dye is a reporter dye (6-carboxyfluorescein) and the other is a quencher dye (6-carboxy-tetramethylrhodamine). When the probe is intact, fluorescence energy transfer occurs and the fluorescent emission from the reporter dye is absorbed by the quencher dye. During the extension phase of the PCR cycle, the fluorescent hybridization probe is cleaved by the 5'-3' nucleolytic activity of the DNA polymerase. Upon cleavage of the probe, the reporter dye emission is no longer effectively transferred to the quencher dye, resulting in an increase in the reporter dye fluorescence emission spectrum. The amount of nucleic acid in a sample can be quantified using any nucleic acid quantification method, including real-time methods or single-point detection methods. Detection can be accomplished in a number of different ways (e.g. staining, hybridization with labeled probes; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P-labeled deoxynucleoside triphosphates such as dCTP or dATP into amplified fragments), it can also be accomplished by any other suitable detection method known in the art for the quantification of nucleic acids. Quantification may or may not include an amplification step.

In some embodiments, the present invention provides markers for identifying and quantifying ligated DNA fragments. In some cases, the ligated DNA fragments can be labeled to aid in downstream applications, such as array hybridization. For example, the ligated DNA fragments can be labeled using random priming methods or nick translation.

A variety of labels (eg, reporter molecules) can be used to label the nucleotide sequences described herein, including but not limited to during amplification steps. Suitable labels include radionuclides, enzymes, fluorescers, chemiluminescent or chromogenic agents, as well as ligands, cofactors, inhibitors, magnetic particles and the like. Examples of such markers are listed in U.S. Patent No. U.S. Patent No. 3,817,837; U.S. Patent No. 3,850,752; U.S. Patent No. 3,939,350; U.S. Patent No. 3,996,345; U.S. Patent No. 4,277,437; Incorporated by reference.

Additional markers include, but are not limited to, β-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, β-glucuronidase, exo-glucanase, and glucoamylase . Fluorescent labels can also be used, as well as specially synthesized fluorescent reagents with specific chemical properties. Fluorescence can be measured in a number of ways. For example, some fluorescent labels undergo changes in excitation or emission spectra, some undergo resonance energy transfer when a fluorescent reporter loses fluorescence, some lose (quench) fluorescence or fluoresce when fluorescence is regained, and some reporter rotation sports.

In addition, multiplex amplifications can be combined rather than increasing the number of amplification cycles per reaction in order to obtain sufficient material for labeling. Alternatively, labeled nucleotides can be incorporated in the final multiple cycles of the amplification reaction, for example 30 PCR cycles (without label) + 10 PCR cycles (with label).

In particular embodiments, the invention provides probes capable of attaching to ligated DNA fragments. As used herein, the term "probe" refers to a molecule (such as an oligonucleotide, whether naturally occurring in a purified restriction digest) capable of hybridizing to another target molecule (such as another oligonucleotide). or produced synthetically, recombinantly or by PCR amplification). When the probe is an oligonucleotide, it can be single-stranded or double-stranded. Probes are very useful in detecting, identifying and isolating specific targets such as gene sequences. In some cases, the probe can be associated with a label such that it is detectable in any detection system, including but not limited to enzymatic (e.g. ELISA, and enzyme-based histochemical assays), fluorescent, radioactive and luminous system.

With respect to arrays and microarrays, the term "probe" is used to refer to any hybridizable material that is immobilized to the array for the detection of nucleotide sequences to which the probes have hybridized. In some cases, the probe can be about 10 bp to 500 bp, about 10 bp to 250 bp, about 20 bp to 250 bp, about 20 bp to 200 bp, about 25 bp to 200 bp, about 25 bp to 100 bp, about 30 bp to 100 bp, or about 30 bp to 80 bp. In some cases, the probe length can be greater than about 10 bp, about 20 bp, about 30 bp, about 40 bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp, about 150 bp, about 200 bp, about 250 bp, about 300bp, about 400bp or about 500bp. For example, the probe can be about 20 to about 50 bp in length. Examples and principles for probe design can be found in WO95/11995, EP 717,113 and WO97/29212.

In some cases, one or more probes can be designed so that they hybridize near sites digested by restriction endonucleases. For example, the probe can be at about 10 bp, about 20 bp, about 30 bp, about 40 bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp, about 150 bp, about Within 200bp, about 250bp, about 300bp, about 400bp, or about 500bp.

In other cases, about 10 bp, about 20 bp, about 30 bp, about 40 bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp, Within about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 400 bp, or about 500 bp, a single, unique probe is designed. Design the probes so that they hybridize on either side of the restriction endonuclease digestion site. For example, a single probe on each side of the primary restriction enzyme recognition site can be used.

In further cases, 2, 3, 4, 5, 6, 7, 8 or more probes can be designed on each side of the restriction endonuclease recognition site, recognizing The loci can then be used to study the same ligation event. For example, 2 or 3 probes can be designed on each side of the restriction endonuclease recognition site. In some embodiments, multiple (2, 3, 4, 5, 6, 7, or 8 or more) probes may be used per primary restriction enzyme recognition site, This can be advantageous in reducing the problem of getting false negative results from a single probe.

As used herein, the term "probe set" refers to a set or group of probes that can interact with one or more primary restriction enzyme recognition sites in the genome for a primary restriction enzyme hybridize.

In some cases, a set of probes may be sequentially complementary to nucleic acid sequences adjacent to one or more primary restriction enzyme recognition sites of a restriction enzyme in genomic DNA. For example, the probe set can be sequentially complementary to nucleotides adjacent to one or more primary restriction endonuclease recognition sites in genomic DNA, these nucleotides are about 10bp to 500bp, about 10bp to 250bp, about 20bp to 250bp , about 20bp to 200bp, about 25bp to 200bp, about 25bp to 100bp, about 30bp to 100bp, or about 30bp to 80bp. The probe set may be complementary to sequences on one side (eg, on either side) or both sides of the restriction endonuclease recognition site. Accordingly, the probes may be complementary to nucleic acid sequences adjacent to each side of one or more primary restriction endonuclease recognition sites in the adjacent genomic DNA. In addition, the probe set may be complementary to a nucleic acid sequence from one or more primary restriction enzyme recognition sites in the genome that is less than about 10 bp, about 20bp, about 30bp, about 40bp, about 50bp, about 60bp, about 70bp, about 80bp, about 90bp, about 100bp, about 150bp, about 200bp, about 250bp, about 300bp, about 400bp, or about 500bp.

In some cases, two or more probes can be designed to hybridize to sequences adjacent to one or more restriction endonuclease recognition sites in the genomic DNA. Probes may overlap or partially overlap.

Probes, probe arrays or probe sets may be immobilized on a support. Supports such as solid supports can be made from a variety of materials - for example glass, silica, plastic, nylon or nitrocellulose. The carrier is preferably rigid and has a planar surface. A vector may have from about 1 to 10,000,000 defined resolved sites. For example, the carrier may have about 10 to 10,000,000, about 10 to 5,000,000, about 100 to 5,000,000, about 100 to 4,000,000, about 1,000 to 4,000,000, about 1,000 to 3,000,000, about 10,000 to 3,000,000, about 10,000 2,000,000, about 100,000 to 2,000,000, or about 100,000 to 1,000,000 identified resolved sites. The density of defined resolved sites can be at least about 10, about 100, about 1000, about 10,000, about 100,000, or about 1,000,000 defined resolved sites per square centimeter. In some cases, each resolved site can be occupied by greater than 95% of a single type of oligonucleotide. In other cases, each resolved site may be occupied by a pooled mixture or set of probes. In further instances, some resolved sites are occupied by pooled mixtures or probe sets of probes, while other resolved sites are occupied by greater than 95% of a single type of oligonucleotide.

In some cases, the number of probes for a given nucleotide sequence on an array may be in large excess relative to the DNA sample to be hybridized to the array. For example, the array can have about 10 times, about 100 times, about 1000 times, about 10,000 times, about 100,000 times, about 1,000,000 times, about 10,000,000 times, or about 100,000,000 times more probes than the amount of DNA in the input sample number.

In some cases, the array can have about 10, about 100, about 1000, about 10,000, about 100,000, about 1,000,000, about 10,000,000, about 100,000,000, or about 1,000,000,000 probes.

Arrays of probes or sets of probes can be synthesized on a support in a stepwise fashion, or can be ligated in presynthesized form. One synthesis method is VLSIPS ^™ (as described in US Patent No. 5,143,854 and European Patent EP 476,014), which requires the use of light to guide the synthesis of oligonucleotide probes in high-density, miniaturized arrays. Algorithms for designing masks to reduce the number of synthesis cycles are described in US Patent No. 5,571,639 and US Patent No. 5,593,839. Arrays can also be synthesized in a combinatorial manner, as described in European patent EP 624,059, by transporting the monomers to the grooves of the carrier through mechanically constrained flow channels. Arrays can also be synthesized by dropping reagents onto supports using an inkjet printer (see eg European Patent EP 728,520).

In some embodiments, the invention provides methods for hybridizing ligated DNA fragments onto an array. A "substrate" or "array" is a collection of purposely created nucleic acids that can be prepared synthetically or biosynthetically and that can be prepared in a number of different ways (e.g., libraries of soluble molecules; attached to resin beads, silicon chips, or other solid Libraries of oligonucleotides with phase vectors) are screened for biological activity. Furthermore, the term "array" includes those nucleic acid libraries made by spotting nucleic acids of substantially any length (eg, from 1 to about 1000 nucleotide monomers in length) on a substrate.

Array technology and various related techniques and applications are described extensively in many textbooks and literature. These include, for example, Lemieux et al., 1998, Molecular Breeding 4, 277-289; Schena and Davis, Parallel Analysis with Biological Chips. in PCR Methods Manual (eds. M. Innis, D. Gelfand, J. Sninsky); DNA Microarrays: A Schena and Davis, 1999, Genes, Genomes and Chips. in Practical Approach (edited by M. Schena) (Oxford University Press, Oxford, UK, 1999); The Chipping Forecast (Nature Genetics special issue; January 1999 supplement); Mark Schena (editor), Microarray Biochip Technology, (Eaton Publishing); Cortes, 2000, The Scientist 14[17]:25; Gwynn and Page, Microarray analysis: the next revolution inmolecular biology, Science, 6 August 1999; and Eakins and Chu, 1999, Trends in Biotechnology, 17, 217-218.

In general, any library can be arranged in an ordered fashion in an array by spatially separating the members of the library. Examples of suitable libraries for arraying include nucleic acid libraries (including libraries of DNA, cDNA, oligonucleotides, etc.), peptide, polypeptide and protein libraries, in addition to libraries comprising any molecule, such as a ligand library .

Libraries can be immobilized or immobilized to a solid support (eg, a solid substrate) to limit diffusion and mixing of members. In some cases, libraries of DNA-binding ligands can be prepared. In particular, libraries can be immobilized to substantially planar solid phases, including membranes and non-porous substrates such as plastic and glass. In addition, libraries can be arranged in a manner that facilitates indexing (ie, referencing or accessing specific members). In some embodiments, members of a library can be applied in spots in a grid formation. Common analysis systems are suitable for this purpose. For example, an array can be immobilized to the surface of a microplate as multiple members in a well, or as a single member in each well. Furthermore, the solid substrate may be a membrane, such as nitrocellulose or nylon membranes (such as are used in blotting assays). Alternative substrates include glass or silicon based substrates. Thus, the library may be immobilized by any suitable method known in the art, for example by charge interaction or by chemical coupling to the walls and bottom of the well or to the surface of the membrane. Other means of alignment and fixation can be used, such as pipetting, dripping, piezoelectric means, inkjet and bubble jet techniques, electrostatic applications, and the like. In the case of silicon-based substrates, libraries can be arrayed and immobilized on the chip using photolithography.

Libraries can be arrayed by being "spotted" onto a solid substrate; this can be done manually or by depositing members using robotics. In general, arrays can be described as macroarrays or microarrays, the difference being the size of the spots. Macroarrays can contain spot sizes of approximately 300 microns or greater and can be easily imaged with existing gel and blot scanners. The size of spots in a microarray can be less than 200 microns in diameter and these arrays often contain thousands of spots. Therefore, microarrays may require specialized robotics and imaging instruments, which require customization. In the review Cortese, 2000, The Scientist 14[11]:26, the instrument is generally described.

Techniques for generating immobilized libraries of DNA molecules are described in the art. In general, most prior art methods describe how to synthesize libraries of single-stranded nucleic acid molecules using, for example, masking techniques to construct various sequence arrangements at discrete locations on a solid substrate. US Patent No. 5,837,832 describes an improved method for producing DNA arrays immobilized to silicon substrates based on very large scale integration technology. In particular, U.S. Patent No. 5,837,832 describes a strategy called "tiling" to synthesize specific probe sets at spatially defined locations on a substrate, which can be used to generate the immobilized DNA of the present invention. library. US Patent No. 5,837,832 also provides citations to earlier techniques that may also have been used. In other cases, arrays can also be constructed using photodeposition chemistry.

Arrays of peptides (or peptidomimetics) can also be synthesized on the surface in such a way that each distinct library member (eg, a unique peptide sequence) is placed at a discrete, predetermined location in the array. The identity of each library member is determined by its spatial position in the array. The location of a binding interaction between a predetermined molecule (eg, a target or probe) and a reaction library member is determined, thereby identifying the sequence of the reaction library member based on the spatial location. These methods are described in the following documents: U.S. Patent No. 5,143,854; WO90/15070 and WO92/10092; Fodor et al., (1991) Science, 251:767; Dower and Fodor, (1991) Ann.Rep.Med.Chem., 26:271.

To facilitate detection, a label (as discussed above) can be used—eg, any readily detectable reporter molecule, such as a fluorescent reporter, bioluminescent reporter, phosphorescent reporter, radioactive reporter, etc. reporter molecule. These reporter molecules, their detection, binding to targets/probes, etc. are discussed elsewhere in this disclosure. Labeling of probes and targets is also disclosed in Shalon et al., 1996, Genome Res 6(7):639-45.

Table 1 below gives examples of some commercial microarray formats (see also Marshall and Hodgson, 1998, Nature Biotechnology, 16(1), 27-31).

Table 1

Table 1 - continued

To generate data from an array-based analysis, a signal indicative of the presence or absence of hybridization between a probe and a nucleotide sequence can be detected. In addition, direct or indirect labeling techniques can also be utilized. For example, direct labeling incorporates fluorescent dyes directly into nucleotide sequences that hybridize to probe-associated arrays (e.g., dye incorporation into nucleotides by enzymatic synthesis in the presence of labeled nucleotides or PCR primers sequence). For example, by using a family of fluorescent dyes with similar chemical structures and properties, direct labeling can generate strong hybridization signals and is easy to implement. Where direct labeling of nucleic acids is involved, cyanine or Alexa analogs can be used in multiplex fluorescence comparison array assays. In other embodiments, direct labeling methods can be used to incorporate epitopes into nucleic acids either before or after hybridization to microarray probes. One or more staining methods and reagents can be used to label the hybridized complex (eg, a fluorescent molecule that binds to an epitope, thereby providing a fluorescent signal via binding of the dye molecule to the epitope of the hybridized population.)

In various embodiments, suitable sequencing methods described herein or known in the art will be used to obtain sequence information from nucleic acid molecules in a sample. Sequencing can be accomplished by classical Sanger sequencing methods known in the art. Sequencing can also be accomplished using high-throughput systems, some of which allow sequenced nucleotides to be detected immediately after or during their incorporation into a growing strand, ie, sequence detection in real-time or substantially real-time. In some instances, the high throughput sequencing generates at least 1,000, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 100,000, or at least 500,000 sequence reads per hour while the sequencing reads can be at least about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 150, about 180, about 210 per read about 240 about 270 about 300 about 350 about 400 about 450 about 500 about 600 about 700 about 800 about 900 or about 1000 bases base.

In some embodiments, high-throughput sequencing involves the use of technologies achievable by Illumina's Genome Analyzer IIX, MiSeq Personal Sequencer, or HiSeq systems, such as those using HiSeq 2500, HiSeq 1500, HiSeq 2000, or HiSeq 1000 machines. These machines use terminator-based sequencing that is reversible by synthetic chemistry. These machines can complete 200 billion DNA reads or more in eight days. Smaller systems are available for runs of 3, 2, 1 day or less.

In some embodiments, high-throughput sequencing involves techniques achievable using the ABI Solid System. This genetic analysis platform enables massively parallel sequencing of clonally amplified DNA fragments attached to beads. The sequencing method is based on sequential ligation with dye-labeled oligonucleotides.

Next-generation sequencing can include ion semiconductor sequencing (eg, using technology from Life Technologies (IonTorrent)). Ion semiconductor sequencing can take advantage of the release of ions when nucleotides are incorporated into DNA strands. For ion semiconductor sequencing, high density arrays of micromechanical wells can be formed. Each well can hold a single DNA template. Below the holes may be an ion-sensitive layer, and below the ion-sensitive layer may be an ion sensor. When nucleotides are added to DNA, H+ is released, which is a measure of the change in pH. The H+ ions are converted into an electrical voltage and recorded by a semiconductor sensor. The array chip can be filled sequentially with nucleotides one after the other. No scans, lights or cameras are required. In some cases, nucleic acids are sequenced using an IONPROTON ^™ sequencer. In some cases, an IONPGM ^™ sequencer was used. Ion Torrent Personal Genome Sequencer (PGM). PGM can complete 10 million reads in two hours.

In some embodiments, high-throughput sequencing involves the use of technologies provided by Helicos BioSciences Corporation (Cambridge, Massachusetts), such as the Single Molecule Sequencing by Synthesis (SMSS) method. SMSS is unique because it allows the entire human genome to be sequenced in up to 24 hours. Finally, SMSS is described in part in US Published Application Nos. 20060024711; 20060024678; 20060012793; 20060012784; and 20050100932.

In some embodiments, high-throughput sequencing involves the use of technologies available through 454 Lifesciences, Inc. (Branford, Connecticut), such as the PicoTiterPlate device, which includes a fiber optic panel that transmits the chemiluminescent signal generated by the sequencing reaction to a The CCD camera in the instrument records. The use of this fiber optic allows detection of a minimum of 20 million base pairs in 4.5 hours.

Methods using bead amplification prior to detection of optical fibers are described in Marguiles, M. et al., "Genome sequencing in microfabricated high-density pricolitre reactors", Nature, doi: 10.1038/nature03959; and U.S. Published Application No. 20020012930; 20030068629; 20030100102; 20030148344; 20040248161; 20050079510, 20050124022; and 20060078909.

In some embodiments, high-throughput sequencing is performed using a Clonal Single Molecule Array (Solexa Corporation) or sequencing-by-synthesis using reversible terminator chemistry. These techniques are described in part in: US Patent Nos. 6,969,488; 6,897,023; 6,833,246; 6,787,308; and US Application Publication Nos. 20040106110;

Next generation sequencing technologies may include Pacific Biosciences' Real Time Technology (SMRT ^™ ). In SMRT, each of the four DNA bases can be attached to one of four different fluorescent dyes. These dyes can be phosphate-linked. A single DNA polymerase can be immobilized with a single molecule of template single-stranded DNA at the bottom of a zero-mode waveguide (ZMW). The ZMW can be a confinement structure that enables the incorporation of a single nucleotide by DNA polymerase to be observed against the background of a fluorescent nucleotide that rapidly diffuses into and out of the ZMW (measured in microseconds). ). It can take milliseconds to incorporate nucleotides into a growing chain. During this time, the fluorescent label can be excited and generate a fluorescent signal, and the fluorescent tag can be cleaved away. The ZMW can be illuminated from below. Attenuated light from the excitation light penetrates the lower 20-30 nm of each ZMW. A microscope can be created with a detection limit of 20 liters (10" liters). The extremely small detection volume enables a 1000-fold improvement in background noise reduction. Detection of the corresponding dye fluorescence indicates which bases are incorporated. The process can be repeated .

In some cases, the next generation sequencing is nanopore sequencing (see eg, Soni GV and Meller A. (2007) Clin Chem 53:1996-2001). A nanopore may be a small hole with a diameter up to about one nanometer. Immersing the nanopore in a conductive liquid and applying an electric current through it creates a tiny current due to the conduction of ions through the nanopore. The amount of current flowing is sensitive to the size of the nanopore. When a DNA molecule passes through a nanopore, each nucleotide on the DNA molecule can clog the nanopore to varying degrees. Thus, as a DNA molecule travels through the nanopore, a change in current passing through the nanopore can represent a readout of the DNA sequence. Nanopore sequencing technology is available from Oxford Nanopore Technologies; eg the GridlON system. A single nanopore can be inserted into a polymer membrane that spans the top of the microwell. Each microwell can have an electrode for a single sensing. The microwells can be fabricated as array chips with 100,000 or more microwells per chip (e.g., more than 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 indivual). Instruments (or nodes) can be used to analyze chips. Data can be analyzed in real time. One or more instruments can be operated at a time. The nanopore may be a protein nanopore, eg a protein alpha-hemolysin, a heptameric protein pore. The nanopores may be nanopores made in a solid state, such as nanometer-sized pores formed in synthetic membranes such as SiNx or SiO2. A nanopore may be a hybrid pore (eg, a protein pore integrated in a solid-state membrane). Nanopores can be nanopores with integrated sensors (e.g. tunnel electrode detectors, capacitive detectors or graphene-based nanopores or edge state detectors (see e.g. Garaj et al., (2010) Nature vol. 67, doi:10.1038/nature09379 )). Nanopores can be functionalized to analyze specific types of molecules (eg DNA, RNA or proteins). Nanopore sequencing can include "strand sequencing," in which intact DNA polymers can pass through a protein nanopore and be sequenced in real time as the DNA translocates through the pore. The enzyme separates the strands of double-stranded DNA and passes the strands through the nanopore. DNA can have a hairpin at one end, and the system can read both strands. In some instances, nanopore sequencing is "exonuclease sequencing," in which individual nucleotides are cleaved from a DNA strand by a processing exonuclease and the nucleotides are passed through the protein nanopore. Nucleotides can transiently bind to molecules (eg, cyclodextrins) within the pores. Characteristic current interruptions can be used to identify bases.

Nanosequencing technology from GENIA is available. Engineered protein pores can be embedded in lipid bilayer membranes. "Active control" techniques can be used to efficiently assemble nanoporous membranes and control the movement of DNA through the channels. In some cases, nanopore sequencing technology was obtained from NABsys. Genomic DNA can be fragmented into strands with an average length of about 100 kb. The 100 kb fragment can be made single stranded and subsequently hybridized with a 6 base probe. Genomic fragments with probes can be driven through the nanopore, which can generate a current trace versus time. Current tracing provides the position of the probes on each genomic fragment. Genomic fragments can be aligned to create probe maps for the genome. This method can be used in parallel for probe library completion. A probe map can be generated for the genome length of each probe. Errors can be corrected using a method called "moving window sequencing by hybridization (mwSBH)". In some cases, nanopore sequencing technology was from IBM/Roche. Electron beams can be used to create nanopore-sized openings in microchips. An electric field can be used to pull the DNA through or through the nanopore. DNA transistor devices in nanopores can contain alternating nanoscale metal or dielectric layers. Discrete charges in the DNA backbone can be trapped by the electric field inside the DNA nanopore. Turning on or off the gate voltage allows the DNA sequence to be read.

Next-generation sequencing may comprise DNA nanoball sequencing (eg, accomplished by Complete Genomics; see eg, Drmanac et al., (2010) Science 327:78-81). DNA can be isolated, fragmented and size selected. For example, DNA can be fragmented (eg, by sonication) to an average length of about 500 bp. Adapters (Adl) can be used to join the ends of the fragments. Adapters can be used to hybridize to anchors for sequencing reactions. DNA with adapters ligated to each end can be PCR amplified. The linker sequence can be modified so that the complementary single-stranded ends join each other to form circular DNA. DNA can be methylated to protect it from cleavage by IIS-type restriction enzymes used in subsequent steps. A linker (eg, a right linker) can have a restriction recognition site that can remain unmethylated. The unmethylated restriction recognition site in the linker can be recognized by a restriction enzyme such as Acul, which can cut the DNA 13 bp to the right of the right linker to form a linear double-stranded DNA. A second set of right and left adapters (Ad2) can be ligated to either end of the linear DNA, and the DNA combined with both adapters can be PCR amplified (eg, by PCR). The Ad2 sequences can be modified so that they bind to each other and form circular DNA. DNA can be methylated, but the restriction enzyme recognition site on the left Adl linker remains unmethylated. A restriction endonuclease (such as Acul) can be used, and the DNA can be cut 13 bp to the left of Adl to form a linear DNA fragment. The third set of right and left adapters (Ad3) can be ligated to the right and left flanks of linear DNA, and the resulting fragments can be amplified by PCR. Linkers can be modified so that they bind to each other and form circular DNA. A type III restriction enzyme (eg EcoP15) can be added; EcoP15 can cut DNA 26 bp to the left of Ad3 and 26 bp to the right of Ad2. This cleavage removes large fragments of DNA and linearizes the DNA again. A fourth set of right and left adapters (Ad4) can be ligated to DNA, amplified (eg, by PCR), and modified so that they bind to each other and form a fully circular DNA template.

Small fragments of DNA can be amplified using rolling circle replication (eg, using Phi 29 DNA polymerase). The four linker sequences can contain hybridizable palindromic sequences, and the single strand can fold upon itself to form DNA nanoballs (DNBs) with an average diameter of about 200-300 nanometers. DNA nanospheres can be attached (eg by adsorption) to the microarray (sequencing flow cell). Flow cells can be silicon wafers and photoresists coated with silicon dioxide, titanium, and hexamethyldisilazane (HMDS). Sequencing can be performed by off-chain sequencing by attaching fluorescent probes to DNA. The fluorescent color of the detected position can be observed by a high-resolution camera. The identity of the nucleotide sequences between the linker sequences can be determined.

In some embodiments, high-throughput sequencing can be performed using AnyDot. Chips (Genovoxx, Germany). In particular, AnyDot. Chips enhance the detection of nucleotide fluorescence signals by 10 to 50 times. AnyDot. Chips and methods of using chips are described in part in International Published Application Nos. WO 02088382, WO 03020968, WO 03031947, WO 2005044836, PCT/EP 05/05657, PCT/EP 05/05655; and German Patents Application numbers DE 101 49 786, DE 102 14 395, DE 103 56 837, DE 10 2004 009 704, DE 10 2004 025 696, DE 10 2004025 746, DE 10 2004 025 694, DE 10 2004 50 4025 025 , DE 10 2004025 745 and DE 10 2005 012 301.

Other high-throughput sequencing systems include those disclosed in Venter, J., et al., Science, Feb. 16, 2001; Adams, M. et al., Science, Mar. 24, 2000; and M.J. Levene et al., Science 299:682-686, January 2003; and US Published Application Nos. 20030044781h and 2006/0078937. In general, such systems involve sequencing target molecules with multiple bases by temporally adding bases through assayed polymerization reactions on nucleic acid molecules, ie, tracking the activity of nucleic acid polymerases on template nucleic acid molecules to be sequenced in real time. The sequence can then be deduced by identifying, at each step, the bases that are catalytically incorporated by the nucleic acid polymerase into the growing complementary strand of the target nucleic acid in the order of base addition. The polymerase on the target nucleic acid molecule complex is located at a position suitable for movement along the target nucleic acid molecule and extension of the oligonucleotide primer at the active site. Multiple labeled classes of nucleotide analogs are located proximate to the active site, each distinguishable class of nucleotide analogs being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid chain is extended by adding a nucleotide analog to the nucleic acid chain at the active site, which is complementary to the nucleotides of the target nucleic acid at the active site, by using a polymerase. As a result of the polymerization step, nucleotide analogs added to the oligonucleotide primers are identified. The steps of providing labeled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated to further extend the nucleic acid strand and determine the sequence of the target nucleic acid.

In particular embodiments, the invention further provides kits comprising one or more components of the invention. The kit can be used for any application obvious to those skilled in the art, including those described above. A kit can comprise, for example, a plurality of association molecules, fixatives, restriction enzymes, ligases, and/or combinations thereof. In some cases, an association molecule can be a protein, including, for example, histones. In some cases, the fixative can be formaldehyde or any other DNA cross-linking agent.

In some cases, the kit can further comprise a plurality of beads. Beads can be paramagnetic and/or coated with capture agents. For example beads can be coated with streptavidin and/or antibodies.

In some cases, kits can include adapter oligonucleotides and/or sequencing primers. In addition, the kit can comprise a device capable of amplifying read pairs using adapter oligonucleotides and/or sequencing primers.

In some cases, the kit may also contain other reagents including, but not limited to, lysis buffers, ligation reagents (such as dNTPs, polymerases, polynucleotide kinases, and/or ligase buffers, etc.), and PCR reagents (such as dNTPs, polymerase and/or PCR buffer, etc.).

The kit may also comprise instructions for using the kit components and/or for generating read pairs.

The computer system 500 shown in FIG. 8 can be understood as a logical device capable of reading instructions from a medium 511 and/or a network port 505, which can optionally be connected to a server 509 with a fixed medium . A system such as that shown in FIG. 8 may include a central processing unit 501 , a disk drive 503 , optional input devices such as a keyboard 515 and/or mouse 516 , and an optional display 507 . Data can be communicated to a local or remote server through the illustrated communication media. Communication media may include any means of transmitting and/or receiving data. For example, a communication medium can be a network connection, a wireless connection or an Internet connection. This connection can provide communication over the world wide web. It is contemplated that, as shown in FIG. 8, data related to the present invention may be transmitted via these networks or connections for receipt and/or comment by a party.

FIG. 9 is a block diagram representing a first example architecture of a computer system 100 that may be used in conjunction with example embodiments of the present invention. As shown in FIG. 9, the example computer system may include a processor 102 for processing instructions. Non-limiting examples of processors include: Intel Xeon™ processor, AMD Opteron™ processor, Samsung 32-bit RISC ARM1176JZ(F)-S v1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8 Apple A4™ processor processor, Marvell PXA 930TM processor, or functionally equivalent processor. Multithreaded execution is available for parallel processing. In some embodiments, multi-processors and multi-core processors may also be used, whether in a single computer system, in a cluster, or distributed across a network of systems comprising multiple computers, cell phones, and/or personal Data assistant device.

As shown in FIG. 9 , a cache memory 104 may be connected to or incorporated in the processor 102 to provide high-speed memory for instructions or data most recently or frequently used by the processor 102 . Processor 102 is connected to Northbridge 106 via processor bus 108 . Northbridge 106 is connected to random access memory (RAM) 110 through memory bus 112 and manages access to RAM 110 through processor 102 . Northbridge 106 is also connected to Southbridge 114 through chipset bus 116 . South bridge 114 is in turn connected to peripheral bus 118 . A peripheral bus may be, for example, PCI, PCI-X, PCI Express, or other peripheral bus. The Northbridge and Southbridge are commonly referred to as the processor chipset and manage the transfer of data between the processor, RAM and peripheral components on the peripheral bus 118 . In some alternative architectures, the Northbridge functionality can be integrated into the processor instead of using a separate Northbridge chip.

In some embodiments, the system 100 may include an accelerator card 122 connected to the peripheral bus 118 . Accelerators may include field programmable gate arrays (FPGAs) or other hardware used to accelerate a process. For example, accelerators can be used for adaptive data reconstruction, or for evaluating algebraic expressions used in extended set processing.

Software and data are stored in external memory 124 and may be stored in RAM 110 and/or cache memory 104 for use by the processor. System 100 includes an operating system for managing system resources; non-limiting examples of operating systems include: Linux, Windows ^™ , MACOS ^™ , BlackBerry OS ^™ , iOS ^™ , and other functionally equivalent operating systems, as well as Application software running on the , used to manage stored data and optimize according to embodiments of the present invention.

In this embodiment, system 100 also includes network interface cards (NICs) 120 and 121 to connect to the peripheral bus to provide a network interface to external storage, such as network attached storage (NAS) and other computers that can be used for distributed parallel processing system.

Figure 10 diagrammatically shows a network 200 having multiple computer systems 202a and 202b, multiple cell phones and personal data assistants 202c, and network attached storage (NAS) 204a and 204b. In an example embodiment, systems 202a, 202b, and 202c can manage data storage and optimize data access to data stored in network attached storage (NAS) 204a and 204b. A data model can be applied to the data and evaluated using distributed parallel processing through computer systems 202c and 202b and cell phone and personal data assistant system 202c. Computer systems 202c and 202b and cell phone and personal data assistant system 202c may also provide parallel processing for adaptive data reconstruction of data stored in network attached storage (NAS) 204a and 204b. Figure 10 shows an example only, and various other computer architectures and systems may be used in conjunction with various embodiments of the invention. For example, blade servers can be used to provide parallel processing. Processor blades can be connected through the backplane to provide parallel processing. Storage can also be attached to the backplane or as network-attached storage (NAS) via a separate network interface.

In some example embodiments, a processor may maintain a separate memory space and transmit data through a network interface, backplane, or other connector for parallel processing by other processors. In other implementations, some or all of the processors may use a shared virtual address storage space.

11 is a block diagram of a multiprocessor computer system 300 using a shared virtual address storage space according to an example embodiment. The system includes a plurality of processors 302a - f that have access to a shared memory subsystem 304 . The system incorporates multiple programmable hardware memory algorithm processors (MAPs) 306a - f into the memory subsystem 304 . Each MAP 306a-f may include memory 308a-f and one or more field programmable gate arrays (FPGAs) 310a-f. MAPs provide configurable functional units, and specific algorithms or portions of algorithms can be provided to FPGAs 310a-f for processing in close cooperation with the respective processors. For example, MAP can be used to evaluate algebraic representations about data models and to perform adaptive data reconstruction in example embodiments. In this example, all processors used for these purposes have global access to each MAP. In one configuration, each MAP may access an associated memory 308a-f using direct memory access (DMA), allowing it to perform tasks independently of, and asynchronously with, a corresponding microprocessor 302a-f. In this configuration, MAPs can directly feed results back to further MAPs for pipeline and parallel execution of algorithms.

The above computer architectures and systems are examples only, and a variety of other computer, cell phone, and personal data assistant architectures and systems can be used in conjunction with the example embodiments, including systems using any combination of: general-purpose processors, co-processors, FPGAs, and Other programmable logic devices, systems on chip (SOCs), application specific integrated circuits (ASICs), and other processing and logic elements. In some embodiments, all or part of a computer system may run in software or hardware. A variety of data storage media may be used in conjunction with example embodiments, including random access memory, hard drives, flash memory, tape drives, disk arrays, network attached storage (NAS), and other local or distributed data storage devices and systems.

In an example embodiment, the computer system may operate using software modules executing on any of the above or other computer architectures and systems. In other embodiments, system functionality may run partially or completely in firmware, programmable logic devices, system-on-chips (SOCs), application-specific integrated circuits (ASICs), or other processing or logic elements, such as The Field Programmable Gate Array (FPGA) shown in Figure 11. The group processor and optimizer can be hardware accelerated, for example by using a hardware accelerator card, such as accelerator card 122 shown in FIG. 9 .

The following examples are intended to illustrate but not limit the invention. These examples are representative of those that may be used, and other procedures known to those skilled in the art may alternatively be used.

Example

Example 1. Method for generating chromatin in vitro

Two pathways for remodeling chromatin deserve special attention: one uses the random deposition of ATP-independent histones onto DNA, and the other uses the ATP-dependent assembly of periodic nucleosomes. The invention allows for the use of either approach in conjunction with one or more of the methods disclosed herein. Examples of two pathways for generating chromatin can be found in Lusser et al. ("Strategies for the reconstitution of chromatin", Nature Methods (2004), 1(1):19-26), the entire contents of which are incorporated herein by reference, Include references cited therein.

Example 2. Genome Assembly Using HI-C Based Technology

Genomes from human subjects were fragmented into pseudocontigs of size 500 kb. Generate multiple read pairs by probing the physical layout of chromosomes in living cells using a Hi-C-based approach. A variety of Hi-C based methods can be used to generate read pairs, including the method shown below: Lieberman-Aiden et al. ("Comprehensive mapping of longrange interactions reveals folding principles of the human genome", Science (2009), 326(5950) :289-293), the entire contents of which are incorporated herein by reference, including the references cited therein. Read pairs were mapped to all pseudo-contigs, and those that mapped to two independent pseudo-contigs were used to build an adjacency matrix based on the mapping data. About 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the read pairs are weighted by taking a function of the distance of the reads to the pseudo-contig edge to mathematically The above embodies the empirically known higher probability of short contacts than long contacts. Then, for each pseudo-contig, the adjacency matrix is analyzed to determine a path through the pseudo-contig by finding a single best-neighboring pseudo-contig, which is determined by having the highest weighted sum. By implementing these methods, it was found that greater than 97% of all pseudocontigs identified their correct neighbor pseudocontigs. Additional experiments can be performed to test the effect of shorter contigs and alternative weighting and path finding methods.

Alternatively, genome assembly using Hi-C data may include computational methods that exploit signals of genomic proximity in Hi-C data sets for ultralong scaffolding for de novo genome assembly. Examples of such computational methods that can be used in conjunction with the methods disclosed herein include the linking adjacent chromatin method proposed by Burton et al. (Nature Biotechnology 31:1119-1125 (2013)); the DNA triangulation method proposed by Kaplan et al. (Nature Biotechnology 31:1143- 47 (2013)), the entire contents of which and any references cited therein are incorporated herein by reference. Furthermore, it should be understood that these computational methods can be used in combination, including with other genome assembly methods shown herein.

For example, the Burton et al.-based method for linking adjacent chromatin can be used in conjunction with the methods disclosed herein, which includes the steps of: (a) aggregating contigs into chromosome sets, (b) mapping contigs within one or more chromosome sets , and (c) assign a relative orientation to each contig. For step (a), contigs were placed into groups using hierarchical clustering. A graph is created where each node initially represents a contig and each edge between nodes has a weight equal to the number of Hi-C read pairs connecting the two contigs. Using hierarchical agglomerative clustering with an average connectivity metric, contigs were mixed until the number of groups was reduced to the desired number of distinct chromosomes (only groups with more than one contig were counted). Non-clustering repeat contigs (the average junction density of this contig with other contigs, normalized by the number of restriction fragment sites, is two times greater than the average junction density) and having too few restriction fragment sites contigs. After clustering, however, each of these contigs was assigned to a group if its average connection density with that group was four times greater than its average connection density with other groups. For step (b), create a graph similar to the clustering step, but edges between nodes are weighted equal to the inverse of the number of Hi-C connections between contigs, normalized by the number of restriction fragment sites in each contig change. Short contigs were excluded from the plot. Compute the minimum spanning tree for this icon. The longest path "backbone" in this tree is found. This spanning tree is then modified to lengthen the trunk by adding to it contigs adjacent to the trunk so that the total edge weights remain tentatively low. After each group discovers the extended backbone, the extended backbone is converted to fully ordered as described below. The backbone is removed from the spanning tree, leaving a set of "branches" containing all contigs not within the backbone. These branches are reinserted into the trunk, longest branches first, with insertion sites chosen so as to maximize the number of contiguous connections in the ordering. Short fragments were not inserted; therefore, multiple small contigs that were clustered did not participate in the final assembly. For step (c), the orientation of each contig within its sequence was determined by considering the exact position of the Hi-C junction alignment on each contig. The probability of assuming a Hi-C join to join two reads at a genomic distance x is roughly 1/x for x > ~100Kb. A weighted directed acyclic graph (WDAG) is created representing all possible ways of orienting contigs in a predetermined order. Each edge in WDAG corresponds to a pair of adjacent contigs along one of its four possible combined directions, and the edge weight is set to the pair observing the set of Hi-C connection distances between the two contigs likelihood, assuming they are immediately adjacent in a given direction. For each contig, the quality score for its orientation was calculated as follows. The log-likelihood of the observed set of Hi-C connections between this contig in its current orientation and its neighboring contigs is found. The contigs were then reversed and the log-likelihood calculated again. Due to the way the direction is calculated, the first log-likelihood is guaranteed to be high. The difference between the log-likelihoods is taken as a quality score.

Alternative DNA triangulation methods like Kaplan et al. can also be used in the methods disclosed herein to assemble genomes from contigs and read pairs. DNA triangulation is based on the use of high-throughput in vivo genome-wide chromatin interaction data to infer genomic positions. For DNA triangulation, CTR types were first quantified by grouping genes into 100-kb bins, each bin representing a large virtual contig, and calculating the average interaction frequency of each placed contig with each chromosome. To assess mapping over long distances, the interaction data of the contig and the 1 mb adjoining on each side were ignored. The average interaction frequency largely separates interactions within chromosomes from interactions between chromosomes and is highly predictive of which chromosome a contig belongs to. Then, a simple multilevel model, the Naive Bayes classifier, was employed to predict the chromosomes of each contig based on their average interaction frequency with each chromosome. The assembled portion of the genome was used to fit a probabilistic one-parameter exponential decay model (domain-driven design (DDD) model) describing the relationship between Hi-C interaction frequency and genomic distance. In each round, contigs were removed from the chromosome, along with 1 Mb of flanking regions on each side. The most probable position of each contig was then estimated based on the interaction case and the decay model. The forecast error is quantified as the absolute value of the distance between the predicted position and the actual position.

The predictability of each contig can be further improved by combining DNA triangulation with long-insert libraries. By knowing the chromosome assignments and the approximate location of each contig, the computational complexity of long-insert scaffolds can be greatly reduced, as each contig only needs to be paired with its neighboring contigs; thus resolving ambiguous contig connections and reducing Assembly errors in which contigs located in distant regions of the chromosome or on different chromosomes are incorrectly linked.

Example 3. Method for haplotype phasing

Because read pairs generated by the methods disclosed herein typically originate from intrachromosomal contacts, any read pair containing a heterozygous site will also carry information related to its phasing. Using this information, reliable phasing at short, medium and even long (megabase) distances can be performed quickly and accurately. Experiments designed for phasing data from one of the 1000 genome triplets (collection of maternal/paternal/offspring genomes) reliably infer phasing. Furthermore, haplotype reconstruction using proximity linkage similar to Selvaraj et al. (Nature Biotechnology 31:1111-1118 (2013)) can also be used in conjunction with the haplotype phasing method disclosed herein.

For example, haplotype reconstruction based on proximity joining methods can also be used in the methods disclosed herein for phased genomes. Haplotype reconstruction using a proximity-joining-based approach combining proximity-joining and DNA sequencing with a probabilistic algorithm for haplotype assembly. First, use chromosome capture technology, such as Hi-C technology, to perform proximity junction sequencing. These methods capture DNA fragments from two distant genomic locations that loop together in three dimensions. After shotgun DNA sequencing of the resulting DNA library, the paired-end sequencing reads have an "insert size" ranging from a few hundred base pairs to tens of millions of base pairs. Thus, short DNA fragments generated in Hi-C assays generate small haplotype blocks, and long fragments eventually join these small blocks together. With sufficient sequencing range, the method could potentially connect variants in non-contiguous blocks and compose each such block into a single haplotype. This data is then combined with a probabilistic algorithm for haplotypes. The probabilistic algorithm utilizes a graph in which nodes correspond to heterozygous variants and edges correspond to overlapping sequence segments of connectable variants. The plot may contain spurious edges caused by sequencing errors or trans interactions. A max-cut algorithm is then used to predict the reduced solution that is most consistent with the haplotype information provided by the set of input sequencing reads. Because the graphs generated by proximity ligation are larger than conventional genome sequencing or paired sequencing, the computation time and number of iterations were modified so that haplotypes could be predicted with reasonable speed and high accuracy. The resulting data can then be used to guide local phasing using Beagle software and sequencing data from genome projects to generate chromosome-spanning haplotypes with high resolution and accuracy.

Example 4. Methods for Metagenome Assembly

Microorganisms are collected from the environment and fixed with a fixative, such as formaldehyde, to form crosslinks within the microbial cells. By using high-throughput sequencing, multiple contigs were generated from microorganisms. Generate multiple read pairs by using Hi-C based techniques. Read pairs that map to different contigs indicate which contigs are from the same species.

Example 5. Method of making very long-range read pairs

DNA was extracted up to a fragment size of 150 kbp using a commercially available kit. DNA was assembled into reconstituted chromatin structures in vitro using commercial kits from Activ Motif. Chromatin was biotinylated, fixed with formaldehyde, and immobilized on streptavidin beads. Digest DNA fragments with restriction enzymes and incubate overnight. The resulting cohesive ends were filled in with α-thio-dGTP and biotinylated dCTP to generate blunt ends. The blunt ends were ligated with T4 ligase. Reconstituted chromatin is digested with proteases to recover ligated DNA. DNA was extracted from beads and subjected to exonuclease digestion to remove biotin from unligated ends. The recovered DNA was sheared and the ends filled in with dNTPs. The biotinylated fragment was purified by pull down with streptavidin beads. In some cases, adapters were ligated and the fragments were PCR amplified for high-throughput sequencing.

Example 6. Method for generating high-quality human genome assemblies

Knowing that the present invention can generate read pairs spanning considerable genetic distances, the utilization of this information for genome assembly can be tested. The present invention can greatly improve possible de novo assembly and attachment of chromosome-length scaffolds. It can be assessed how completely an assembly can be produced and how much data is required to use the present invention. To assess the efficacy of this method to generate data useful for assembly, standard Illumina shotgun and XLRP libraries were constructed and sequenced. In one case, data from 1 Illumina HiSeq lane each of the standard shotgun library and data from the XLRP library were used. The data generated by each method was examined and compared to various existing assembly software. Optionally, new software specifically adapted to the unique data generated by the present invention is also documented. Optionally, use well-characterized human samples as a reference to compare assemblies produced by the method to assess its accuracy and completeness. Using the knowledge gained from the previous analysis, assembly software was generated to increase the efficient and effective use of XLRP and shotgun data. Using the methods described herein, the quality of the resulting genome assembly was comparable to, or better than, the December 2002 draft mouse genome.

One sample that can be used for this analysis is NA12878. DNA from sample cells is extracted using a variety of published techniques for maximizing DNA fragment length. Standard Illumina TruSeq shotgun libraries and XLRP libraries were constructed separately. Each library yields a single HiSeq lane of 2x150bp sequences, yielding approximately 150 million read pairs per library. Shotgun data were assembled into contigs using a whole genome assembly algorithm. Examples of these algorithms include: Meraculous as described in Chapman et al. (PLOS ONE6(8):e2350(2011)) or standard genetics as described in Simpson et al. (Genome research22(3):549–56 (2012)) algorithm. Align the XLRP library reads to the contigs generated by the initial assembly. Alignment was used to further link contigs. Once the effectiveness of XLRP for joining contigs is established, Meraculous assembly can be extended to simultaneously integrate shotgun and XLRP libraries into a single assembly process. Meraculous lays a solid foundation for assembling software, optionally producing integrated assembling software to meet the specific needs of the invention. The assembled human genome of the present invention is compared to any known sequence to assess the quality in the genome assembly.

Example 7. Method for phasing heterozygous SNPs in human samples with high accuracy from small data sets

In one experiment, approximately 44% of the heterozygous variants in the test human sample data set were phased. Capture all or nearly all phased variants within one read length of the restriction site. By using in silico analysis, more variants can be captured for phasing by using longer read lengths and using a combination of restriction enzyme(s) for digestion. Using a combination of restriction enzymes with different restriction sites increases the proportion of the genome (and thus heterozygous sites) within one of the two restriction sites participating in each read pair. In silico analysis showed that using various combinations of the two restriction enzymes, the method of the present invention could phase over 95% of the known heterozygous positions. Additional enzymes and larger read lengths further increase the fraction of heterozygous sites that are observed and phased, up to complete coverage and phasing.

The hybrid site coverage achievable with various combinations of the two restriction enzymes was calculated. The first three combinations were tested with the pipeline for heterozygous sites in read proximity. For each of these combinations, an XLRP library was generated and sequenced. The resulting reads are aligned to the human reference genome and compared to the known haplotypes of the samples to determine the accuracy of the experimental procedure. Phase up to 90% or more of heterozygous SNPs in human samples with 99% or better accuracy using only 1 lane of Illumina HiSeq data. Additionally, variants were further captured by increasing the read length to 300bp. The read region around the observable restriction site is effectively doubled. Apply additional restriction enzyme combinations to increase coverage and accuracy.

Example 8. Extraction and influence of high molecular weight DNA:

DNA up to 150kbp was extracted using commercially available kits. Figure 7 demonstrates that an XLRP library can be generated from captured read pairs up to the maximum fragment length of the extracted DNA. Accordingly, the methods disclosed herein are expected to be able to generate read pairs from even longer stretches of DNA. There are many well-established methods for the recovery of high molecular weight DNA, and these methods can be used in conjunction with the methods or protocols disclosed herein. Extraction methods are used to generate large fragment lengths of DNA from which XLRP libraries are created and the resulting read pairs can be evaluated. For example, large molecular weight DNA can be extracted by the following methods: (1) mild cell lysis, according to Teague et al (Proc. (11): e1000711(2009)); and (2) agarose gel plug, according to Wing et al. References are incorporated herein, including any references cited therein, or by using the Aurora System from Boreal Genomics. These methods are capable of generating longer DNA fragments than are typically required for next-generation sequencing; however, other suitable methods known in the art may be substituted to achieve similar results. The Aurora System provides unexpected results and can isolate and concentrate DNA from tissue or other prepared samples to lengths up to or exceeding a million bases. DNA extracts were prepared using each of these methods, starting from a single GM12878 cell culture to control for possible differences in sample levels. The size distribution of the fragments was assessed by pulsed field gel electrophoresis according to Herschleb et al. (Nature Protocols 2(3):677-84 (2007)). Using the methods described above, extremely long strings of DNA can be extracted and used to construct XLRP libraries. The XLRP library was then sequenced and aligned. The resulting read data were analyzed by comparing the genomic distance between the read pairs and the fragment sizes observed from the gel.

Example 9. Reduction of read pairs from undesired genomic regions

RNA complementary to undesired genomic regions is generated by in vitro transcription and incorporated into reconstituted chromatin prior to crosslinking. When the complementary RNA binds to one or more undesired genomic regions, RNA binding reduces the efficiency of crosslinking at these regions. Thereby reducing the abundance of DNA from these regions in the cross-linked complex. Recombined chromatin was biotinylated and immobilized, and used as described above. In some cases, RNAs are designed to target repetitive regions in the genome.

Example 10. Increasing read pairs from desired chromatin regions

DNA from desired chromatin regions is produced in double-stranded form for gene assembly or haplotype analysis. Representation of DNA from undesired regions is correspondingly reduced. Double-stranded DNA from desired chromatin regions is generated by laying primers across these regions at multiple kilobase spacing. In other embodiments of the method, the blanket spacing is varied to achieve desired replication efficiencies for desired regions of different sizes. Primer binding sites are contacted via the desired region, optionally by melting the DNA. A new strand of DNA is synthesized using the laid primers. Undesired regions are reduced or eliminated, for example, by targeting these regions with single-stranded DNA-specific endonucleases. The remaining desired regions are optionally amplified. Prepared samples were processed using sequencing library preparation methods described elsewhere herein. In some embodiments, read pairs spanning up to the length of each desired chromatin region are generated from each of these desired chromatin regions.

While preferred embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that these Examples are by way of illustration only. Various modifications, changes and substitutions will occur to those skilled in the art without departing from the scope of the present invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that structures and methods within the scope of such claims and their equivalents be covered by such claims. It is intended that the following claims define the scope of the invention and that structures and methods within the scope of such claims and their equivalents be covered by these claims.

Claims (22)

1. a kind of method for genome assembling, including：

Obtain multiple contigs；

Reconstruct chromatin is obtained, the reconstruct chromatin includes the naked DNA compound with the nucleoprotein detached；

It is reconstructed in the data that chromatinic physical layout generates from by detecting, generates multiple readings pair；

It is read the multiple to positioning to the multiple contig, so as to generate read location data；

The contig is arranged using the read location data；And

Determine the path by the contig, the order and/or the side towards genome that the path represents the contig To.

2. according to the method described in claim 1, wherein the multiple contig is by using the generation of air gun PCR sequencing PCR, the bird Rifle PCR sequencing PCR includes：

Make subject's DNA break of long section into the uncertain random fragment of size；

The segment is sequenced with high-flux sequence method, to generate multiple sequencing reads；With

The sequencing read is assembled, to form multiple contigs.

3. the method according to claim 1 or claim 2 wherein by using the technology included the following steps, passes through Detection reconstructs chromatinic physical layout, generates the multiple reading pair：

Reconstruct chromatin is made to be crosslinked with fixative, to form DNA- protein cross objects；

With the crosslinked DNA- protein of one or more restriction enzyme cleavages, to generate multiple DNA- containing cohesive end Protein complex；

The cohesive end described in the nucleotide filling-in containing one or more markers, generating flat end then makes blunt end cloning Together；

The multiple DNA protein complexes is made to be fractured into segment；

By using one or more markers, the segment containing contact is pulled down；With

The segment containing contact is sequenced with high-flux sequence method, to generate multiple readings pair.

4. according to the method described in claim 1, wherein the multiple read to being to reconstruct chromatinic physical layout by detecting Generation, the reconstruct chromatin is to be answered by will be obtained from the naked DNA of one or more subject's samples with the histone detached Close what is formed.

5. according to the method described in claim 1, wherein for the multiple reading pair, by using the read to the overlapping The function of the distance at group edge, the reading pair of weighting at least 80% embody short contact probability more higher than long contact.

6. according to the method described in claim 1, wherein the method provides the genome assembling of human experimenter, wherein from people The DNA of class subject generates multiple contigs, and wherein reconstructs chromatin by using as made from subject's naked DNA, raw Into multiple readings pair.

7. according to the method described in claim 1, wherein the method further includes：

Identify it is the multiple read to one or more of heterozygous sites；With

Reading pair of the identification containing pairs of heterozygous sites, wherein from the identification of the pairs of heterozygous sites, can determine for equipotential Gene variant determines phase data.

8. according to the method described in claim 1, wherein from single DNA molecules obtain it is the multiple read pair, wherein at least 1% It reads to crossing over at least distance of 30kB on single DNA molecules, and wherein described reading in 14 days to generating.

9. according to the method described in claim 8, wherein at least 10% reading is to crossing on the single DNA molecules extremely The distance of few 50kB.

10. according to the method described in claim 8, wherein at least 1% reading is to crossing on the single DNA molecules extremely The distance of few 100kB.

11. according to the method described in claim 1, wherein described reading in 7 days to generating.

12. according to the method described in claim 1, wherein the multiple reading pair is obtained from single DNA molecules in vitro；Wherein extremely Few 1% reading is to crossing over at least distance of 30kB on the single DNA molecules.

13. according to the method for claim 12, wherein at least 10% reading is to crossing on the single DNA molecules At least distance of 30kB.

14. according to the method for claim 13, wherein at least 1% reading is to crossing on the single DNA molecules At least distance of 50kB.

15. according to the method described in claim 7, wherein the multiple reading pair is obtained from single DNA molecules；Wherein at least 1% The reading to across at least distance of 30kB on the single DNA molecules, and wherein be more than 70% accuracy into Row determines phase data for Alielic variants.

16. according to the method for claim 15, wherein at least 10% reading is to crossing on the single DNA molecules At least distance of 50kB.

17. according to the method for claim 15, wherein at least 1% reading is to crossing on the single DNA molecules At least distance of 100kB.

18. according to claim 15 to 17 any one of them method, wherein being carried out with the accuracy for being more than 90% for equipotential Gene variant determines phase data.

19. according to the method described in claim 7, wherein the multiple reading pair is obtained from single DNA molecules in vitro；Wherein extremely Few 1% reading to across at least distance of 30kB on the single DNA molecules, and wherein be more than 70% it is accurate Degree carries out determining phase data for Alielic variants.

20. according to the method for claim 19, wherein at least 10% reading is to crossing on the single DNA molecules At least distance of 30kB.

21. according to the method for claim 19, wherein at least 1% reading is to crossing on the single DNA molecules At least distance of 50kB.

22. according to claim 19 to 21 any one of them method, wherein being carried out with the accuracy for being more than 90% for equipotential Gene variant determines phase data.